promega caspase-glo 3 7 assay protocol

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promega celltiter-fluor cell viability assay protocol

Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·1.Description ..........................................................................................................12.Product Components and Storage Conditions ............................................43.Reagent Preparation and Storage ...................................................................54.Protocols for the CellTiter-Fluor™ Cell Viability Assay ..........................5A.Determining Assay Sensitivity, Method 1........................................................6B.Determining Assay Sensitivity, Method 2........................................................7C.Example Viability Assay Protocol.....................................................................8D.Example Multiplex Assay Protocol (with luminescent caspase assay).......9E.Recommended Controls (10)5.General Considerations ..................................................................................106.References .........................................................................................................117.Related Products ..............................................................................................121.DescriptionThe CellTiter-Fluor™ Cell Viability Assay (a)is a nonlytic, single-reagent-addition fluorescence assay that measures the relative number of live cells in a culture population after experimental manipulation (Figures 1 and 2). The CellTiter-Fluor™ Cell Viability Assay measures a conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability (1). Results obtained using the CellTiter-Fluor™ Cell Viability Assay correlate well with other established methods of determining cell viability(Figure 3). The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC). The substrate enters intact cells where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells (Figure 4). This live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium.The CellTiter-Fluor™ Cell Viability Assay also can be used in a single-well,sequential, multiplex format with other downstream chemistries to normalize data by cell number. Data from the assay can serve as an internal control andCellTiter-Fluor™ Cell Viability AssayAll technical literature is available on the Internet at: /protocols/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@allow identification of errors resulting from cell clumping or compoundcytotoxicity. The CellTiter-Fluor™ Cell Viability Assay is compatible with most Promega luminescence assays or spectrally distinct fluorescence assay methods,such as assays measuring caspase activation, reporter gene expression or orthogonal measures of viability. However, some P450-Glo™ multiplexprotocols may require removing culture supernatant to a separate assay well before performing the assay because of isoform-specific competitive inhibition of the cytotochrome P450 enzymes by the coumarin product of the CellTiter-Fluor™ Cell Viability Assay reaction.Figure 1. Schematic diagram of the CellTiter-Fluor™ Cell Viability Assay.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6868M ACellTiter-Fluor™ ReagentAdd GF-AFC Substrate to Assay Buffer to create the CellTiter-Fluor™ Reagent.MeasureAssay BenefitsMeasure the Relative Number of Live Cells in Culture: Nonlytic, single-reagent-addition, homogeneous, “add-mix-measure” protocol.Get More Data from Every Well: The CellTiter-Fluor™ Cell Viability Assay can be performed in multiplex with most Promega luminescence assays.Normalize Data for Cell Number: Normalizing data for live-cell number makes results more comparable well-to-well, plate-to-plate, day-to-day.Figure 3. The CellTiter-Fluor™ Cell Viability Assay shows strong correlation with established methods for measuring viability. Panel A. The GF-AFC Substrate signal from serial dilutions of live cells plotted against results from the CellTiter-Glo ®Luminescent Cell Viability Assay (Cat.# G7570), which measures cellular ATP.Panel B.The GF-AFC Substrate signal from serial dilutions of live cells plottedagainst results achieved using the CellTiter-Blue ®Cell Viability Assay (Cat.# G8080).Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6867M AA.B.R e s o r u f i n F l u o r e s c e n c e (R F U )2,5002,6002,7002,8002,9003,0003,100GF-AFC Fluorescence (RFU)A T P -B a s e d A s s a y L u m i n e s c e n c e (R L U )0GF-AFC Fluorescence (RFU)LIVE-CELL SUBSTRATE:cell-permeant fluorogenic substrate for the live-cellprotease (Gly-Phe-AFCoumarin)NucleusLive CellLive-cell protease substrate can cross the cell membrane.Active Live-CellProteaseO CF 3GF–N HOGFOCF 3OH 2N6995M AFigure 2. CellTiter-Fluor™ Cell Viability Assay chemistry. The cell-permeant substrate enters the cell, where it is cleaved by the live-cell protease activity to produce the fluorescent AFC. The live-cell protease is labile in membrane-compromised cells and cannot cleave the substrate.Figure 4. The CellTiter-Fluor™ Cell Viability Assay signal derived from viable cells (untreated) is proportional to cell number. Dead cells (treated) do not contribute appreciable signal in the assay.2.Product Components and Storage ConditionsProductSize Cat.#CellTiter-Fluor™ Cell Viability Assay10mlG6080Cat.# G6080 contains sufficient reagents for 100 assays at 100µl/assay in a 96-well plate format or 400 assays at 25µl/assay in a 384-well plate format. Includes:• 1 × 10ml Assay Buffer• 1 × 10µl GF-AFC Substrate (100mM in DMSO)ProductSize Cat.#CellTiter-Fluor™ Cell Viability Assay5 × 10mlG6081Cat.# G6081 contains sufficient reagents for 500 assays at 100µl/assay in a 96-well plate format or 2,000 assays at 25µl/well in a 384-well format. Includes:• 5 × 10ml Assay Buffer• 5 × 10µl GF-AFC Substrate (100mM in DMSO)ProductSize Cat.#CellTiter-Fluor™ Viability Assay2 × 50mlG6082Cat.# G6082 contains sufficient reagents for 1,000 assays at 100µl/assay in a 96-well plate format or 4,000 assays at 25µl/well in a 384-well format. Includes:• 2 × 50ml Assay Buffer• 2 × 50µl GF-AFC Substrate (100mM in DMSO)Storage Conditions: Store the CellTiter-Fluor™ Cell Viability Assaycomponents at –20°C. See product label for expiration date.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6866M ACells or Cell Equivalents/WellA F C F l u o r e s c e n c e (R F U )3.Reagent Preparation and Storagepletely thaw the CellTiter-Fluor™ Cell Viability Assay components in a37°C water bath. Vortex the GF-AFC substrate to ensure homogeneity, thenbriefly centrifuge for complete substrate volume recovery.2.Transfer the GF-AFC Substrate (10µl for Cat.# G6080 and G6081; 50µl for Cat.#G6082) into the Assay Buffer container (10ml for Cat.# G6080 and G6081; 50mlfor Cat.# G6082) to form a 2X Reagent. Mix by vortexing the contents until thesubstrate is thoroughly dissolved.Note: The solution may initially appear “milky” when the GF-AFC substrate isdelivered to the buffer. This is normal. The substrate will dissolve withvortexing. The CellTiter-Fluor™ Reagent may be scaled to accommodate thevolumes required for downstream multiplexes. To do this, use 1/5 the volumeof buffer when you prepare the reagent (i.e., 10µl of the GF-AFC Substrate in2ml of Assay Buffer). Be sure to label the bottle to indicate that this is a moreconcentrated reagent, suitable for multiplex assays. Add the reagent at 1/5 thevolume of the cell culture.Storage: The CellTiter-Fluor™ Viability Reagent should be used within 24hours if stored at room temperature. Unused GF-AFC Substrate and AssayBuffer can be stored at 4°C for up to 7 days with no appreciable loss ofactivity.4.Protocols for the CellTiter-Fluor™ Cell Viability AssayMaterials to Be Supplied by the User•96-, 384-, or 1536-well opaque-walled tissue culture plates compatible with fluorometer (clear or solid bottom)•multichannel pipettor or liquid-dispensing robot•reagent reservoirs•fluorescence plate reader with filter sets for AFC (380–400nm Ex/505Em)•orbital plate shaker•compound known to cause 100% cytotoxicity or lytic detergent (digitonin, Calbiochem Cat.# 300410 or Sigma-Aldrich Cat.# D141 at 20mg/ml in DMSO).If you have not performed this assay on your cell line previously, werecommend determining assay sensitivity using your cells and one of the two methods described below (Section 4.A or 4.B). If you do not need to determineassay sensitivity for your cells, proceed to Section 4.C.Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6.Dilute digitonin to 300µg/ml in water. Using a multichannel pipet,carefully add 10µl of the diluted digitonin to all wells of columns 7–12 to lyse cells (treated samples). Add 10µl of water to all wells of columns 1–6to normalize the volume (untreated cells).7.Add 100µl of the CellTiter-Fluor™ Reagent to all wells, mix briefly by orbital shaking and incubate at 37°C for at least 30 minutes.Note:Longer incubations may improve assay sensitivity and dynamic range. However, do not incubate longer than 3 hours, and be sure to shieldplates from ambient light.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·8.Measure resulting fluorescence with a fluorometer (380–400nm Ex /505nm Em )Note: You may need to adjust instrument gains (applied photomultiplier tube energy).9.Calculate the practical sensitivity for your cell type by making a signal-to-noise calculation for each dilution of cells (10,000 cells/well; 5,000cells/well; 2,500 cells/well, etc.).Viability S:N =(Average Untreated – Average Treated)Note: The practical level of assay sensitivity for the assay is a signal-to-noise ratio of greater than 3 standard deviations (derived from reference 1).4.B.Determining Assay Sensitivity, Method 21.Harvest adherent cells (by trypsinization, etc.), wash with fresh medium (to remove residual trypsin) and resuspend in fresh medium.Note:For cells growing in suspension, proceed to Step2.2.Determine the number of viable cells by trypan blue exclusion using a hemacytometer, then adjust the cells by dilution to 100,000 viable cells/ml in at least 20ml of fresh medium.Note:Concentrate the cells by centrifuging and removing medium if the pool of cells is less than 100,000 cells/ml.3.Divide the volume of diluted cells into separate tubes. Subject one tube to "moderate" sonication (empirically determined by postsonicationmorphological examination) to rupture cell membrane integrity and to simulate a 100% dead population. The second tube of untreated cells will serve as the maximum viable population.4.Create a spectrum of viability by blending sonicated and untreatedpopulations in 1.5ml microcentrifuge tubes as described in Table 2.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·4.B.Determining Assay Sensitivity, Method 2 (continued)5.After mixing each blend by gently vortexing, pipet 100µl of each blend into8 replicate wells of a 96-well plate. Add the 100% viable cells to column 1,95% viable to column 2, etc. Add cell culture medium only to column 10 toserve as a no-cell control.6.Add CellTiter-Fluor™ Reagent in an equal volume (100µl per well) to allwells, mix briefly by orbital shaking, then incubate for at least 30 minutesat 37°C.Note:Longer incubations may improve assay sensitivity and dynamicrange. However, do not incubate longer than 3 hours, and be sure to shieldplates from ambient light.7.Measure resulting fluorescence with a fluorometer (380–400nm Ex/505nm Em).Note: You may need to adjust instrument gains (applied photomultipliertube energy).8.Calculate the practical sensitivity for your cell type by making a signal-to-noise calculation for each blend of cell viability (X = 95, 90%, etc.).Viability S:N = (Average 100% – Average X%)Standard Deviation of 0% (viable cells)Note: The practical level of assay sensitivity for the assay is a signal-to-noise ratio of greater than 3 standard deviations (derived from reference 1).4.C.Example Viability Assay Protocol1.Set up 96-well assay plates containing cells in culture medium at desireddensity.2.Add test compounds and vehicle controls to appropriate wells so that thefinal volume is 100µl in each well (25µl for a 384-well plate).3.Culture cells for the desired test exposure period.4.Add CellTiter-Fluor™ Reagent in an equal volume (100µl per well) to allwells, mix briefly by orbital shaking, then incubate for at least 30 minutesat 37°C.Note:Longer incubations may improve assay sensitivity and dynamicrange. However, do not incubate more than 3 hours, and be sure to shieldplates from ambient light.5.Measure resulting fluorescence using a fluorometer (380–400nm Ex/505nm Em).Note:You may need to adjust instrument gains (applied photomultipliertube energy).Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·4.D.Example Multiplex Assay Protocol (with luminescent caspase assay)1.Set up 96-well assay plates containing cells in culture medium at the desired density.2.Add test compounds and vehicle controls to appropriate wells so that the final volume is 100µl in each well (25µl for a 384-well plate).3.Culture cells for the desired test exposure period.Note:Caspase activation is a transient event dictated by compound potency and cell cycle susceptibility. Time course experiments are often useful for defining peak caspase activity and cytotoxicity.4.Add 20µl of CellTiter-Fluor™ Reagent (prepared as 10µl substrate in 2ml Assay Buffer) to all wells, and mix briefly by orbital shaking. Incubate for at least 30 minutes at 37°C.Note: Longer incubations may improve assay sensitivity and dynamic range. However, do not incubate longer than 3 hours, and be sure to shield plates from ambient light.5.Measure resulting fluorescence using a fluorometer (380–400nm Ex /505nm Em ).Note: You may need to adjust instrument gains (applied photomultiplier tube energy).6.Add an equal volume of Caspase-Glo ®3/7 Reagent prepared as described in Technical Bulletin #TB323 to wells (100–120µl per well), incubate for 30minutes, then measure luminescence using a luminometer.Figure 5. Multiplex of CellTiter-Fluor™ Assay and Caspase-Glo ®3/7 Assay.The CellTiter-Fluor™ Reagent was added to wells and viability measured after incubation for 30 minutes at 37°C. Caspase-Glo ®3/7 Reagent was added andluminescence measured after a 30-minute incubation (10,000 cells/well).Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6865M A® 3/7 Assaylog 10[paclitaxel] ML u m i n e s c e n c e (R L U )F l u o r e s c e n c e (R F U )4.E.Recommended ControlsNo-Cell Control: Set up triplicate wells without cells to serve as a control to determine background fluorescence.Untreated Cells Control:Set up triplicate wells with untreated cells to serve as a vehicle control. Add the same solvent used to deliver the test compounds to the vehicle control wells.Optional Test Compound Control: Set up triplicate wells without cells but containing the vehicle and test compound to test for possible interference with the assay chemistry.Positive Control for Viability:Set up triplicate wells containing cells treated with a compound known to be toxic to the cells used in your model system.5.General ConsiderationsOptical Filters and Instrumentation:Fluorogenic dyes exhibit distinct absorption (excitation) and emission profiles when a light energy source is applied. Most fluorometers or multimode instruments contain optical band-pass filters that restrict the wavelengths of light used to excite a fluorophore and the wavelengths passing through to the detector. Note that deviation from the optimal filter set recommendations (Figure 6) may adversely affect assay sensitivity and performance.Figure 6. Optimal excitation and emission spectra for AFC.Background Fluorescence and Inherent Serum Activity:Tissue culturemedium that is supplemented with animal serum may contain detectable levels of the protease marker used to measure live-cells. This protease activity may vary among different lots of serum. To correct for variability, determine background fluorescence using samples containing medium plus serum without cells.Temperature: The generation of fluorescent product is proportional to the live-cell protease activity. The activity of this protease is influenced by temperature.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6864M AWavelength (nm)F l u o r e s c e n c e (R F U )For best results, we recommend incubating at a constant controlled temperatureto ensure uniformity across the plate. After adding reagent and briefly mixing,we suggest one of two options:1.At 37°C in a water-jacketed incubation module (Me’Cour, etc.).Note:Incubation at 37°C in a CO2culture cabinet may lead to edge-effectsresulting from thermal gradients.2.At room temperature with or without orbital shaking.Note: Assays performed at room temperature may require more than30minutes of incubation for optimal sensitivity. However, do not incubatelonger than 3 hours.Assay Controls: In addition to a no-cell control to establish background fluorescence, we recommend including a maximum viability (untreated cells)and maximum cytotoxicity control in the experimental design. The maximum viability control is established by adding vehicle only (used to deliver the test compound to test wells). In most cases, this consists of a buffer system ormedium and the equivalent amount of solvent added with the test compound.The maximum cytotoxicity control can be determined using a compound thatcauses 100% cytotoxicity or a lytic compound added to compromise viability (digitonin). See Section 4.A.Viability Marker Half-Life:The activity of the protease marker found has nohalf-life in viable cells. Viable cells will process the substrate to liberate the AFC fluorophore. However, when cells lose membrane integrity, the protease activity declines very quickly. Therefore enzymatic instability of the live-cell proteaseoutside of viable cells establishes GF-AFC as a good marker for cell viability.Light Sensitivity: Although the GF-AFC Substrate demonstrates good general photostability, the liberated AFC fluorophore (after contact with protease) can degrade with prolonged exposure to ambient light sources. We recommend shielding the plates from ambient light at all times.Cell Culture Medium:The GF-AFC Substrate is introduced into the test wellusing an optimized buffer system that mitigates differences in pH fromtreatment. In addition, the buffer system supports protease activity in a host of different culture media with varying osmolarity. With the exception of media formulations with either very high serum content or phenol red indicator, no substantial performance differences will be observed among media.6.References1.Niles, A.L. et al. (2007) A homogeneous assay to measure live and dead cells in thesame sample by detecting different protease markers. Anal. Biochem.366, 197–206.2.Zhang, J-H. et al.(1999) A simple statistical parameter for use in evaluation andvalidation of high-throughput screening assays. J. Biomol. Screen. 4, 67–73.Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·7.Related ProductsCell Viability and CytotoxicityAssays ProductSize Cat.#MultiTox-Fluor Multiplex Cytotoxicity Assay 10ml G9200MultiTox-Glo Multiplex Cytotoxicity Assay 10ml G9270CytoTox-Glo™ Cytotoxicity Assay 10ml G9290CytoTox-Fluor™ Cytotoxicity Assay10ml G9260CellTiter-Glo ®Luminescent Cell Viability Assay 10mlG7570CytoTox-ONE™ Homogeneous Membrane Integrity Assay1,000–4,000 assaysG7891CellTiter-Blue ®Cell Viability Assay20mlG8080Additional Sizes Available.Apoptosis AssaysProductSize Cat.#Caspase-Glo ®2 Assay 10ml G0940Caspase-Glo ®6 Assay 10ml G0970Caspase-Glo ®3/7 Assay 10ml G8091Caspase-Glo ®8 Assay 10ml G8201Caspase-Glo ®9 Assay10ml G8211Apo-ONE ®Homogeneous Caspase 3/7 Assay10ml G7790Additional Sizes Available.Reporter Gene AssaysProductSize Cat.#Bright-Glo™ Luciferase Assay System 10ml E2610Steady-Glo ®Luciferase Assay System10ml E2510Additional Sizes Available.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·(a)Patent Pending.© 2007–2012 Promega Corporation. All Rights Reserved.Apo-ONE, Caspase-Glo, CellTiter-Blue, CellTiter-Glo and Steady Glo are registered trademarks of Promega Corporation.BrightGlo, CellTiter-Fluor, CytoTox-Fluor, CytoTox-Glo, CytoTox-ONE and P450-Glo and are trademarks of Promega Corporation.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.。

Promega--双萤光素酶报告基因

SEAP（分泌性碱性磷酸酶） CAT（氯霉素转乙酰基酶） GFP（绿色荧光蛋白）
•分泌性报告基因 (不需裂解) -真核细胞没有背景表达 -易于使用 -设备现成(液闪仪,层析) •显微镜: 亚细胞定位 •不需底物
萤光素酶报告基因的主要优点
• • • • 非放射性检测快（比CAT等）灵敏度高（可比CAT检测灵敏度高100倍）半衰期短（在理想条件下，可检测到10-20 摩尔的萤光素酶分子。在哺乳动物细胞中半衰期3小时，在植物细胞中半衰期3.5小时。而CAT在哺乳动物细胞中的半衰期约50小时）
•细菌，血清等内源活性高 •需要底物
GUS（葡糖醛酸糖苷酶）
•灵敏度好 •植物中内源活性低
• 90% 的 E.coli, 人／动物细胞中有内源活性 •酶的扩散可引起 “过度报告” •需要底物
•在 HeLa , 癌和其它细胞类型中有高的内源活性 •需要底物 •放射性的 •灵敏度底 •50ｈ半衰期 •背景可能高 •折叠 “情形” 可导致信号差别
荧光计（Fluorometer）
液闪仪（Scintilation Counter）电荷耦合装置光学成像系统（CCD opitical imaging system）
荧光显微镜
各种报告基因的优点和缺点
报告基因萤光素酶优点
•优越的灵敏度 –高信号值 –无内源活性
•灵敏度好
缺点
•需要底物
ß-gal
0.76 0.71 0.79 0.74
第二天处理Ｃ计算如下：
1.00
１
２３
处理D
Δ 活性倍数 (791,021/19,154) =
41,30 55,81
（16,062/21,631)

普洛麦格 caspase-glo 3 7 检测试剂盒 ctb323 中文说明书

2021版 CTB323原英文技术手册TB323中文说明书适用产品目录号： G8090、G8091、G8092和G8093Caspase-Glo ®3/7 Assay普洛麦格(北京)生物技术有限公司Promega (Beijing) Biotech Co., Ltd 地址：北京市东城区北三环东路36号环球贸易中心B座907-909电话：************网址：技术支持电话：800 810 8133(座机拨打)，400 810 8133(手机拨打)技术支持邮箱：*************************CTB3232021制作1所有技术文献的英文原版均可在/ protocols 获得。

请访问该网址以确定您使用的说明书是否为最新版本。

如果您在使用该试剂盒时有任何问题，请与Promega 北京技术服务部联系。

电子邮箱：*************************1. 描述 (2)2. 产品组分和储存条件 (4)3. 试剂制备和储存 (6)4. 细胞检测中Caspase-3和Caspase-7活性的检测 (6)4. A. 检测条件 (7)4. B. 采用96孔板培养的细胞的标准检测方案 (10)4. C. 采用康宁®球形微孔板培养的3D微组织的标准检测方案 (10)5. 采用纯化半胱天冬酶进行Caspase-3或Caspase-7活性检测 (11)5. A. 检测条件 (11)5. B. 半胱天冬酶纯酶的标准检测方案 (12)6. 一般注意事项 (17)7. 相关产品 (19)8. 内容变更总结 (21)Caspase-Glo® 3/7 Assay普洛麦格(北京)生物技术有限公司Promega (Beijing) Biotech Co., Ltd 地址：北京市东城区北三环东路36号环球贸易中心B座907-909电话：************网址：技术支持电话：800 810 8133(座机拨打)，400 810 8133(手机拨打)技术支持邮箱：*************************CTB3232021制作21. 描述Caspase-Glo®3/7检测系统(a,b)是一种用来检测Caspase-3和Caspase-7活性的均相发光检测系统。

3D Cell Analysis说明书

欢迎关注Promega官方微信目录1. 什么是3D细胞培养 (3)2. 3D细胞培养的应用 (4)3. 3D细胞培养的分类 (5)4. 3D培养细胞的检测 (6)1）细胞健康检测 (7)• 细胞活性检测 (8)• 细胞凋亡检测 (10)• 细胞毒性检测 (12)2）代谢检测 (14)• 二核苷酸检测系统 (15)• 能量代谢检测系统 (16)• 氧化应激检测系统 (17)5. 检测仪器 (19)33D 细胞培养是能在细胞培养过程中为细胞提供一个更加接近体内生存条件的微环境的细胞培养技术。

■ 什么是3D 细胞培养?很长一段时间以来，科学家们一直依靠平板培养的2D 细胞来研究细胞和疾病的机制。

2D 细胞模型对于细胞培养和处理当然简单且经济。

然而，我们可以看到在过去的十年里，3D 细胞培养越来越受欢迎，因为它们在生理上更为相关，更能代表体内组织。

仔细思考，我们体内没有一种细胞以独立于其他细胞或组织的形式进行单层生长。

相反，大多数细胞自然存在于复杂的三维结构中，包括细胞外基质中的不同细胞类型。

众多的细胞-细胞和细胞-基质相互作用都对它们的行为有着深刻的影响。

此外，2D 单分子膜可以均匀地获得营养和氧气，而肿瘤等细胞团则不是这样。

3D 肿瘤球体更能代表体内肿瘤，与外层相比，内部细胞获得营养和氧气的机会更少，形成自然梯度。

类器官、球状体和3D 细胞模型研究在包括疾病建模和再生医学在内的许多应用中表现出了巨大的潜力。

相对于2D 模型，类器官和球状体等3D 细胞模型使我们有机会在生理学相关背景下更好地理解生物学的复杂性。

经过验证的实验方案和教育资源增强了我们对于培养和分析类器官和球状体的信心，引领3D 模型取得成功。

Quiescence2, nutrientsand assay reagentsDifferences in Cellular Responses“compound is non -toxic”“compound is toxic”4■ 为何要使用3D 培养细胞模型?监测3D 培养物的生物学变化处理iPS 细胞肿瘤活检建好的普通细胞系分化的iPS 细胞CRISPR 转染支持培养敲除一个蛋白（siRNA ）用蛋白处理表达一个蛋白用miRNA 处理小分子抑制剂物理学变化（如缺氧）可能在治疗前和/或后发生微球体培养3D Culture真皮成纤维细胞细胞工程细胞健康变化代谢变化表达变化基因组分析细胞模型越来越多地被用来了解疾病机制和药物研发治疗。

人半胱氨酸蛋白酶-3(caspase-3)试剂盒使用方法

人半胱氨酸蛋白酶-3（caspase-3）试剂盒使用方法本试剂盒仅供研究使用。

检测范围：96T3 pmol/L -80 pmol/L使用目的：本试剂盒用于测定人血清、血浆及相关液体样本中半胱氨酸蛋白酶-3（caspase-3）含量。

实验原理本试剂盒应用双抗体夹心法测定标本中人半胱氨酸蛋白酶-3（caspase-3）水平。

用纯化的人半胱氨酸蛋白酶-3（caspase-3）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入半胱氨酸蛋白酶-3（caspase-3），再与HRP 标记的半胱氨酸蛋白酶-3（caspase-3）抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB 显色。

TMB 在HRP 酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。

颜色的深浅和样品中的半胱氨酸蛋白酶-3（caspase-3）呈正相关。

用酶标仪在450nm 波长下测定吸光度（OD 值），通过标准曲线计算样品中人半胱氨酸蛋白酶-3（caspase-3）浓度。

试剂盒组成1 30 倍浓缩洗涤液 20ml×1 瓶 7 终止液 6ml×1 瓶2 酶标试剂 6ml×1 瓶 8 标准品（160 pmol/L） 0.5ml×1 瓶3 酶标包被板 12 孔×8 条 9 标准品稀释液 1.5ml×1 瓶4 样品稀释液 6ml×1 瓶 10 说明书 1 份5 显色剂A 液 6ml×1 瓶 11 封板膜 2 张6 显色剂B 液 6ml×1/瓶 12 密封袋 1 个标本要求1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。

若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融2．不能检测含NaN3 的样品，因NaN3 抑制辣根过氧化物酶的（HRP）活性。

操作步骤1. 标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。

Promega 乐灵特r荧光素荧光素试剂说明书

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA1.Description (1)2.Product Components and Storage Conditions (2)3.Principle of the Assay (2)4.Reagent Composition and Testing (2)A.Luciferase/Luciferin (L/L) Reagent (2)B.Reconstitution Buffer (2)5.L/L Reagent Reconstitution (2)6.ATP Assay Procedure (3)A.Luminometer Preparation (3)B.Performing the ATP Assay (3)7.References (5)8.Related Products (5)1.DescriptionThe ENLITEN ®rLuciferase/Luciferin (rL/L) Reagent (a)is intended for the rapid and quantitative detection of adenosine 5´-triphosphate (ATP). The rL/L Reagent is designed to measure 10–11to 10–16mole ATP.Some of the many applications for the ENLITEN ®rL/L Reagent include:•Indirect measurement of bacteria, yeasts, fungi and other microorganisms in foodstuffs, beverages, water, woodpulp, cosmetics and other products.•Assay of enzymes that produce or degrade ATP.•Quantitation of ATP in biological fluids.ENLITEN ®rLuciferase/Luciferin ReagentAll technical literature is available on the Internet at: /tbs/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useofthissystem.E-mail:********************.2.Product Components and Storage ConditionsProduct Size Cat.#ENLITEN ®rLuciferase/Luciferin Reagent100 assays FF2021Components include:•1 vial rLuciferase/Luciferin Reagent •12ml Reconstitution BufferStorage Conditions:Prior to reconstitution, the L/L Reagent andReconstitution Buffer must be stored at –20°C.3.Principle of the AssayRecombinant luciferase catalyzes the following reaction (1):ATP + D -Luciferin + O 2→Oxyluciferin + AMP + Pyrophosphate + CO 2+ Light (560nm)When ATP is the limiting component in the luciferase reaction, the intensity of the emitted light is proportional to ATP concentration. Measurement of thelight intensity using a luminometer permits direct quantitation of ATP (2,3).4.Reagent Composition and Testing4.A.rLuciferase/Luciferin (rL/L) ReagentThe rL/L Reagent is supplied lyophilized; once it has been reconstituted withReconstitution Buffer, it contains purified luciferase, D -luciferin, Tris-acetatebuffer (pH 7.75), ethylenediaminetetraacetic acid (EDTA), magnesium acetate,bovine serum albumin (BSA) and dithiothreitol (DTT). Sodium azide (0.02%) isincluded as a preservative.4.B.Reconstitution BufferThe Reconstitution Buffer contains buffer, salts and other components thatmust be added to the vial of lyophilized rL/L Reagent to reconstitute thereagent.5.rL/L Reagent ReconstitutionATP present in fingerprints, glassware, etc. Do not touch the outside of the gloves with your fingers or skin.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USAbottle labeled Reconstitution Buffer to the vial, recap with the rubber stopper and gently swirl the vial to dissolve the contents. DO NOTshake the dissolved rL/L Reagent. Allow the rL/L Reagent to rehydrate at room temperature for 1hour before using.needed. The activity of the reconstituted rL/L Reagent diminishes roughly 15% after 2 days of storage at 4°C. Be sure to allow the rL/L Reagent to return to room temperature prior to use.If long-term storage is needed, the reconstituted rL/L Reagent can be stored in single-use aliquots at –20°C. Avoid multiple freeze-thaws. The activity of the reconstituted rL/L Reagent diminishes by roughly 50% after two weeks at–20°C.6.ATP Assay Procedure6.A.Luminometer PreparationLight output from the L/L reaction is usually measured in a luminometer.Refer to the instruction manual of your luminometer for proper instrumentsetup and operation. Proper care of your luminometer is important for lowassay “background” and precision in ATP measurements. If your instrument has a reagent delivery system, it is essential that the reagent injectors andsupply tubing are kept clean and aseptic. If the instrument manual does not contain instructions for the cleaning and maintenance of the injectors, contact the manufacturer for instructions. After cleaning the injectors, rinse the system by priming 10 times with sterile, distilled or deionized water. Be sure tocarefully rinse any filters on the reagent tubing as well.6.B.Performing the ATP AssayOnce reconstituted, the rL/L Reagent is sufficient for 100 ATP assays,assuming 0.1ml reagent per assay. Refer to your luminometer instrumentmanual for the recommended procedure for ATP assays. The rLuciferase/Luciferin Reagent has been designed for use in most luminometer protocols.Use disposable pipette tips when adding rL/L Reagent, sample, buffer/water, extractant or ATP standard; use a new tip for each addition. Pipet gently toavoid generating aerosols that could contaminate assay reagents.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA6.B.Performing the ATP Assay (continued)A “blank” containing rL/L Reagent and the sample buffer and extractant usedto prepare your samples should be run in the assay to determine the amountof “background” relative light units (RLU) to be subtracted from the sampleRLU. Sample RLU values should be corrected for possible buffer/extractantinhibition of light output when converting RLU to ATP mass. This is done byconstructing an ATP standard curve (Figure 1) using an appropriate volume ofbuffer/extractant instead of water to dilute the ATP standard. We recommendthat samples and the ATP standard curve are performed in at least duplicatefor accurate measurement (3). The percent coefficient of variation (standarddeviation divided by the mean RLU times 100) for each set of measurementsshould be 10% or less over the entire assay range.Note:A new ATP standard curve must be made fresh daily or whenever anew aliquot of rL/L Reagent is used.Figure 1. A representative ATP standard curve obtained using the ENLITEN ®rL/L Reagent and a Berthold Luminometer Model 9501.Ten microliters of ATP standard diluted to the proper concentrations was added to a cuvette and assayed using rL/L Reagent according to the luminometer protocol. A 1-second delay time after rL/L Reagent injection and 10-second RLU signal integration time were used.This ATP standard curve is for illustration purposes only ; it is important to Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA7.References1.DeLuca, M.A and McElroy, W.D. (1978) Purification and properties of fireflyluciferase. Methods Enzymol.57, 3–15.2.McElroy, W.D. and DeLuca, M.A. (1983) Firefly and bacterial luminescence: Basicscience and applications. J. Appl. Biochem.5, 197–209.3.Lundin, A. and Thore, A. (1975) Analytical information obtainable by evaluation ofthe time course of firefly bioluminescence in the assay of ATP. Anal. Biochem.66,47–63.8.Related ProductsProduct Size Cat.#GloMax™ 20/20 Luminometer 1 instrument E5311GloMax™ 20/20 Luminometer with Single Auto-Injector 1 instrument E5321GloMax™ 20/20 Luminometer with Dual Auto-Injector 1 instrument E5331GloMax™ 96 Microplate Luminometer 1 each E6501GloMax™ 96 Microplate Luminometer with Single Reagent Injector 1 each E6511GloMax™ 96 Microplate Luminometer with Dual Reagent Injectors 1 eachE6521Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA(a)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.© 1993, 2001, 2005, 2007, 2009 Promega Corporation. All Rights Reserved.ENLITEN is a registered trademark of Promega Corporation.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.。

Caspase 3 活性检测试剂盒(比色法)

Caspase 3 活性检测试剂盒(比色法)产品简介：Caspase(Cysteine-requiring Aspartate Protease)家族在介导细胞凋亡过程的起着极其重要的作用，其成员包括Caspase1~11等,均属于蛋白酶家族。

Caspase 3又称CPP32、Yama、apopain、Caspase-3，属于CED-3亚家族，是细胞凋亡过程中的一个关键酶。

Caspase 3 可以剪切procaspase 2、6、7和9，可直接特异性剪切许多Caspase底物（如PARP、ICAD等），并在细胞核凋亡过程中起到重要作用。

Jimei Caspase 3活性检测试剂盒（比色法）(Caspase 3 Colorimetric A ssay Kit)的检测原理是利用Caspase 3催化底物acetyl-Asp-Glu-Val-Asp p-nitroanilide(Ac-DEVD-pNA)产生黄色的游离硝基笨胺pNA (p-nitroaniline)，通过测定分光光度比色法测定pNA在400~410nm处吸光值，从而间接获得Caspase 3的活性。

自备材料：1、水浴锅或恒温箱2、96孔板3、酶标仪或分光光度操作步骤（仅供参考）：1、制备标准曲线：①按照Caspase Lysis buffer：Assay buffer=1：9的比例配制适量的标准品稀释液。

②用标准品稀释液稀释pNA(10mM)，使pNA分别达到200μM、100μM、50μM、25μM、12.5μM、6.25μM，另外设置一般不加pNA仅含标准品稀释液作为零管，把以上系列浓度物质作为标准品。

③取pNA浓度分别为200μM、100μM、50μM、25μM、12.5μM、6.25μM、0的标准品各100μl加入至96孔板或取适量体积加入至容量不超过100μl的比色杯，测定405nm处吸光值即A405。

④用每一个标准品的A405 减去不含pNA的空白对照的A405，计算出实际的吸光值。

Caspase

Caspase生化特性caspase家族属于半胱氨酸蛋白酶，一般在细胞内以无活性酶形式存在；当其作用于特异性底物，产生异二聚体，同时发出光子。

Caspase检测凋亡的原理通过检测细胞内caspase酶活性了解细胞的凋亡功能。

试剂渗透进入细胞后，使细胞内caspase 酶释放，作用于其特异性底物，产生发光体或荧光，发光酶（荧光酶）作用于发光体，产生光。

发光度与细胞内caspase酶活性成正比。

Caspase酶活性反映了细胞内凋亡途径激活情况，即细胞的凋亡能力。

如何做对照组？阴性对照：caspase抑制剂和培养基；或者只加完全培养基。

试剂盒没有caspase抑制剂？不必要。

Caspase试剂盒组成：caspase3/7特异性底物；含发光酶，细胞溶解液的缓冲液。

Caspase试剂盒分类：caspase3/7,caspase8,caspase9.(后两个是内源性凋亡途径参与物）试剂盒的选择：一般细胞凋亡检测选用caspase3/7试剂盒，如果用于分析凋亡上游启动子，根据对凋亡途径（内源性/外源性）的兴趣，选择caspase8/caspase9试剂盒即可。

Caspase3/7介绍Caspase3/7是一种发光分析法，可用于检测粘附细胞或悬浮生长细胞内caspase3/7的酶活性。

试剂盒中包含了发光前体caspase3/7的Z-DEVD-aminoluciferin 冻干底物，DEVD为抑制剂，保证了试剂盒的特异性。

底物被切割后产生发光体，发光体作为发光酶的底物与之反应后，发出光。

Caspase3/7试剂已经对caspase酶活力，发光酶活力以及细胞溶解做了优化。

将试剂以混合物形式加入样本，导致细胞溶解，底物的切割和产生流性光信号。

可用于研究细胞凋亡和凋亡抑制。

实验大概需用2小时一轮。

检测波长一般检测发光体没有内置的波长选择功能，都采用全部可见光检测。

原因有二：1.不需用滤光；2.滤光后敏感度降低。

内源性凋亡途径：病毒，紫外线或线粒体胞膜破坏，细胞色素C释放至胞液等因素导致内源性凋亡途径激活，从而产生含有caspase-9前体的凋亡小体复合物，复合物导致caspase-9活化，接着caspase-9使下游介导凋亡的死亡效应caspase酶活化，包括caspase3,caspase6,caspase7。

Caspase-3活性测定试剂盒

允许适当调整样品加样量)
北京索莱宝科技有限公司
反应缓冲液μl
无样品空白对照
95
待测样品μl
－
底物 μl
5
总体积μl
100
高酶活性样品 85 10 5 100
低酶活性样品 60 35 5 100
产品说明： 1、Caspase酶活力单位定义：参考 One unit is the amount of enzyme that will cleave 1.0 nmol of the colorimetric pNA-substrate per hour at 37 ºC under saturated substrate concentrations。即当底物饱和时,一个Caspase酶活力单位定义为37 ºC 1小时水解pNA底物，产生1nmol游离pNA。根据pNA标准曲线和样品OD值，可计算出Caspase酶活力单位。 2、除了处理组外，可设置阳性凋亡诱导剂组，阴性实验对照可包括不具有凋亡诱导作用的试剂(如果能得到)处理组、溶剂对照组、或凋亡诱导零时间点。
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北京索莱宝科技有限公司
×105个细胞或2个孔的12孔板细胞。勿用丝氨酸蛋白酶抑制剂如E-64和leupeptin以免抑制Caspase活性。 1、（1）细胞裂解：1000g 5分钟收集细胞，吸尽上清。每2~10×106细胞加50~100µl裂解液震荡裂解，冰浴 10分钟，再次震荡。
(2) 组织裂解：3~10mg组织加100 µl裂解液放入小型玻璃匀浆器，上下匀浆30次。转移匀浆液于离心管。 2、4 ºC 12,000g 10分钟，取上清测定，或-70 ºC保存。 3、蛋白定量：用Bradford法测定蛋白浓度。裂解液含还原剂不宜采用BCA法。应使蛋白浓度达到1-3 mg/ml，相当于每10 μl待测样品含10-30μg蛋白，否则应增加细胞用量。

双荧光素酶报告基因分析promega

双荧光素酶报告基因分析1. 介绍荧光素酶报告基因表达的转录调控常被用来研究培养细胞的生物学特性。

荧光素酶是理想的报告基因，因为哺乳动物细胞中不含内源性荧光素酶，一旦转录完成立刻就生成功能性的荧光素酶。

Dual-Luciferase®双荧光素酶报告基因检测系统中含有在同一细胞中同时表达的两种荧光素酶。

通常，报告基因实验中往往会受到各种实验条件的影响，共转染的“对照”报告基因会作为内对照，为试验提供一基准线。

实验报告基因经过内参照的处理可以减小细胞活性和转染效率对实验的影响，因此双报告系统减少了外部干扰，使得实验数据更可信。

实验中报告基因和对照基因的酶没有种源同源性，萤火虫荧光素酶和海肾荧光素酶对应不同的反应底物，反应中没有任何的交叉干扰。

萤火虫荧光素酶底物和海肾荧光素酶底物分别与检测试剂反应可以使灵敏度最大化。

由于超强的光信号和超高的信噪比，本系统被广泛用于制药和生物技术产业中。

双荧光素酶报告基因检测系统适配于各种培养哺乳细胞的培养基，如1640，MEM，DMEM，F12等。

这些试剂与被动裂解液所附带的试剂盒，可以从Promega试剂盒中分开，单独使用。

具有超高灵敏度和超宽线性范围的Veritas™微孔板发光检测仪特别适合DLR 报告基因检测系统，Veritas™软件中预装了DLR 的检测程序使得安装更为方便，内置自动加样器使得应用更为简单。

Veritas™微孔板发光检测仪使用荧光素酶检测试剂II (LAR II)最低可以检测到1X10-19 mol 荧光素酶分子，使用Stop & Glo®试剂可以检测到1X10-18 mol 海肾荧光素酶分子，检测线性范围分别为8 和6 个数量级。

所有的检测均采用纯化的重组萤火虫荧光素酶(E1701)和纯化的重组海肾荧光素酶。

图1－3 使用Promega 公司Dual-Luciferase®双荧光素酶报告基因检测系统，萤火虫荧光素酶(1x10-19 到1x10-11 mol)和海肾荧光素酶(1x10-14 mol)在Modulus™仪上测量结果。

碧波Caspase-3活性分光光度法检测试剂盒

For Research Use Only碧波Caspase-3活性分光光度法检测试剂盒Cat Number：Specification：Store at:4℃for one yearExpire date：MADE BY BIOBOX碧波Caspase-3活性分光光度法检测试剂盒一、产品简介Caspase 3 活性分光光度法检测试剂盒(Caspase 3 Activity Colorimetric Assay Kit)是采用分光光度法检测细胞或组织裂解液中Caspase 3酶活性或纯化的Caspase 3酶活性的试剂盒。

Caspase (Cysteine-requiring Aspartate Protease)是一个在细胞凋亡过程中起重要作用的蛋白酶家族。

Caspase 3也称CPP32、Yama或apopain，有时被写作caspase-3或caspase3，属于caspase家族的CED-3亚家族(CED-3 subfamily)，是细胞凋亡过程中的一个关键酶。

Caspase 3是哺乳动物细胞中研究最多的一个caspase。

Caspase 3可以剪切procaspase 2、6、7和9，并可以直接特异性剪切许多caspase 底物，包括PARP (poly(ADP-ribose) polymerase) ，ICAD (Inhibitor of caspase-activated deoxyribonuclease)，gelsolin和fodrin等。

这些由caspase 3介导的蛋白剪切是细胞凋亡分子机制的重要组成部分。

另外，caspase 3在细胞核凋亡过程中也起到了关键作用，包括染色质固缩(chromatin condensention) ，DNA片段化(DNA fragmentation)等。

同时caspase 3对细胞起泡(Cell blebbing)也起到关键作用。

Caspase-3在正常状态下以酶原的形式存在于胞浆中，没有活性；但在细胞发生凋亡阶段，它被激活，活化的Caspase-3由两个大亚基和两个小亚基组成，裂解相应的胞浆胞核底物，最终导致细胞凋亡。

2021-Promega萤光素酶技术讲座-生物发光技术的前世今生

Promega萤光素酶30周年庆技术讲座季生物发光技术的前世今生目录1.自然界的生物发光与萤光素酶的发现2.萤光素酶从自然界走向实验台3.萤光素酶为何是最理想的报告基因4.生物发光的检测1.自然界的生物发光⚫自然界的发光生物⚫发光生物，为何要发光⚫生物发光机理的探索之路-萤光素酶的发现自然界中的生物发光世界上有很多意想不到的地方都存在会“发光”的生物，比如发光的蘑菇、发光的海洋生物，以及发光的昆虫等，如我们最为熟悉的萤火虫。

发光生物为何要发光人们认为，在生物中产生光的原因根据每个物种不同，其原因各不相同。

目前估计大约有1500种不同的物种可以利用生物化学反应产生光，并且每天还有更多的具有这种能力的物种被发现。

生物发光机理发现约17世纪，爱尔兰化学家、物理学家和自然哲学家Robert Boyle 发现了能够产生微光的腐烂木材，经过研究发现，发光需要氧气的参与。

这是关于生物发光本质的第一份有文献记载的报道。

到20世纪40年代，约翰霍普金斯大学年轻的生物化学教授William McElroy 开始质疑生物发光的分子基础，他知道产生光需要消耗能量，经研究发现ATP 可能是该能量的来源。

他的开创性研究，鼓舞了年轻一代科学家们。

Luciferase 萤光素酶Luciferin萤光素2.萤光素酶从自然界走向实验台⚫萤光素酶商品化产品的诞生⚫萤光素酶发光的原理⚫生物发光与荧光的区别2012新型专利萤光素酶NanoLuc ®诞生2015基于NanoLuc ®萤光素酶的NanoBRET ™技术诞生2016基于NanoLuc ®萤光素酶的NanoBiT ®蛋白互补技术诞生2020基于NanoBiT ®的Lumit ™技术诞生Promega 正式进入免疫检测领域2017基于NanoLuc ®萤光素酶的HiBiT 蛋白标签技术诞生··························································································································································1991第一代萤光素酶产品诞生Luciferase Assay System & pGL2 Vectors1995第一代双萤光素酶检测系统DLR TM 诞生DLR TM & pGL3 Vectors1999UltraGlo ™Recombinant Luciferase 诞生& CellTiter-Glo ®细胞活力检测系统诞生2003Caspase-Glo ®3/7细胞凋亡检测系统诞生2007One-Glo ™Luciferase Assay System 诞生Celebrating 30 Years of Innovation and Discovery Using Bioluminescent TechnologyPromega 萤光素酶技术30年发展里程碑30 Years Of Bioluminescent Technology Innovations ()不同萤光素酶工具与发光机理萤火虫海肾深海细脚刺虾萤光素酶底物萤光素酶萤光素酶底物萤光素酶萤光素酶生物发光与荧光的区别什么是发光？发光是光子（光）发射的过程。

promega 双荧光素酶报告基因检测系统快速Protocol

•••••••••••••••••••ORDERING /TECHNICAL INFORMATION:Reagent Preparation1.1X P L B :Add 1 volume of 55X P a s s i v e L y s i s B u f f e r (P P L B ) to 4 volumes of distilled water. Mix well. Store at 4°C (≤1 month).2.L A R I I : Resuspend the lyophilized LL u c i f e r a s e A s s a y S u b s t r a t e in L L u c i f e r a s e A s s a y B u f f e r I I (10ml for Cat.# E1910, E1960; 105ml for Cat.# E1980). Store at –20°C (≤1 month) or –70°C (≤1 year).3.S t o p & G l o ®R e a g e n t :a.Add 2.1ml of 50X SS t o p & G l o ®S u b s t r a t e to 105ml of S S t o p & G l o ®B u f f e r in the amber S S t o p & G l o ® R e a g e n t bottle provided. Vortex 10 seconds. Store at –20°C for 15 days.b.For a smaller amount of 11X S t o p & G l o ® R e a g e n t :To the required amount of S S t o p & G l o ®B u f f e r , add 50X S t o p & G l o ®S u b s t r a t e to a final 1X concentration. (For example, add 0.2ml of 50X SS t o p & G l o ®S u b s t r a t e to 10ml of SS t o p & G l o ®B u f f e r to make a 1X solution of S S t o p & G l o ® R e a g e n t .)Cell Lysis1.Remove growth media from cultured cells.2.Rinse cultured cells in 1X PBS. Remove all rinse solution.3.Dispense the recommended volume (below) of 11X P L B into each culture vessel.Volumes of 1X PLB to Use in Step 3.Passive Lysis Active Lysis Plate Size 1X PLB Dish/Plate Size 1X PLB 6-well 500µl 100 × 200mm 1ml 12-well 250µl 60 × 15mm 400µl 24-well 100µl 35 × 12mm 200µl 48-well 65µl 6-well 250µl 96-well 20µl 12-well 100µl 4.Passive Lysis :Gently rock/shake the culture vessel for 15 minutes at room temperature. Transfer lysate to a tube or vial.**For automated applications, the DLR™ Assay is performed directly in the multiwell plate.Additional protocol information is available in Technical Manual #TM040 or #TM046, available online at:Cell LysisRemove growth media from cells.Rinse with 1X PBS.Add 11X P L B .Perform lysis.Transfer to a new tube or vial.*2866M A 02_0A•••••••••••••••••••ORDERING /TECHNICAL INFORMATION:Dual-Luciferase ®and Dual-Luciferase ® 1000 Assay ProtocolsAdditional protocol information is available in Technical Manual #TM040 or #TM046, available online at:Plate with ≤20µl of PLB Lysate/well.Dispense 100µl of L A R I I .Measure firefly luciferase activity.Dispense 100µl of S t o p & G l o ®R e a g e n t .Measure Renilla luciferase activity.Assay with Manual or Single-Injector LuminometerPredispense100µl of LL A R I I into luminometer tube.Program luminometer.Transfer 20µl of PLB Lysate. Mix.Measure firefly luciferase activity.Dispense 100µl of S t o p & G l o ®R e a g e n t .Measure Renilla luciferase activity.Assay with 96-Well PlateBefore you begin:Set injectors 1 and 2 to dispense 100µl of L A R I I and S S t o p & G l o ®R e a g e n t , respectively.For measurements, use a 1- to 2-second delay and a 5- to 10-second read time.(inside luminometer)Repeat cycle for remaining wells in plate.2864M A 02_0A2865M A 02_0A。

CellTiter Glo Luminescent Cell Viability Assay Protocol

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·1.Description (1)2.Product Components and Storage Conditions (4)3.Performing the CellTiter-Glo ®Assay (5)A.Reagent Preparation (5)B.Protocol for the Cell Viability Assay (6)C.Protocol for Generating an ATP Standard Curve (optional) (7)4.Appendix (7)A.Overview of the CellTiter-Glo ®Assay..............................................................7B.Additional Considerations..................................................................................8C.References............................................................................................................11D.Related Products. (12)1.DescriptionThe CellTiter-Glo ®Luminescent Cell Viability Assay (a–e)is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The CellTiter-Glo ®Assay is designed for use with multiwell-plate formats, making it ideal for automated high-throughput screening (HTS) and cell proliferation and cytotoxicity assays. The homogeneous assay procedure (Figure 1) involves adding a single reagent (CellTiter-Glo ®Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium or multiple pipetting steps are not required.The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present (Figure 2).The amount of ATP is directly proportional to the number of cells present in culture in agreement with previous reports (1). The CellTiter-Glo ®Assay relies on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase), which generates a stable “glow-type” luminescent signal and improves performance across a wide range of assay conditions. The luciferase reaction for this assay is shown in Figure 3. The half-life of the luminescent signal resulting from this reaction is greater than five hours (Figure 4). This extended half-life eliminates the need for reagent injectors and provides flexibility for continuous or batch-mode processing of multiple plates. The unique homogeneous format reduces pipetting errors that may be introduced during the multiple steps required by other ATP-measurement methods.CellTiter-Glo ®Luminescent Cell Viability AssayAll technical literature is available on the Internet at: /protocols/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useofthissystem.E-mail:********************Figure 1. Flow diagram showing preparation and use of CellTiter-Glo ®Reagent.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3170M A 12_0ACellTiter-Glo CellTiter-Glo MixerLuminometer®System Advantages•Homogeneous:“Add-mix-measure” format reduces the number of plate-handling steps to fewer than that required for similar ATP assays.•Fast:Data can be recorded 10 minutes after adding reagent.•Sensitive:Measures cells at numbers below the detection limits of standard colorimetric and fluorometric assays.•Flexible:Can be used with various multiwell formats. Data can be recorded by luminometer or CCD camera or imaging device.•Robust:Luminescent signal is very stable, with a half-life >5 hours,depending on cell type and culture medium used.•Able to Multiplex:Can be used with reporter gene assays or other cell-based assays from Promega (2,3).Figure 3. The luciferase reaction.Mono-oxygenation of luciferin is catalyzed byluciferase in the presence of Mg 2+, ATP and molecular oxygen.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3171M A 12_0A L u m i n e s c e n c e (R L U )Cells per Well10,00060,00020,00030,00040,00050,0000R² = 0.9990.5 × 1061.0 × 1061.5 × 1062.0 × 1062.5 × 1063.0 × 1063.5 × 1064.0 × 106r² = 0.99020,00010,00030,00040,00050,000r² = 0.9900100200300400HO SN S N O S N S N OCOOH +ATP+O 2Ultra-Glo™ Recombinant Luciferase +AMP+PP i +CO 2+LightBeetle Luciferin OxyluciferinMg 2+0Figure 2. Cell number correlates with luminescent output.A direct relationship exists between luminescence measured with the CellTiter-Glo ®Assay and the number of cells in culture over three orders of magnitude. Serial twofold dilutions of HEK293cells were made in a 96-well plate in DMEM with 10% FBS, and assays wereperformed as described in Section 3.B. Luminescence was recorded 10minutes after reagent addition using a GloMax ®-Multi+ Detection System. Values represent the mean ± S.D. of four replicates for each cell number. The luminescent signal from 50HEK293 cells is greater than three times the background signal from serum-supplemented medium without cells. There is a linear relationship (r 2= 0.99)between the luminescent signal and the number of cells from 0to 50,000 cells per well.Figure 4. Extended luminescent half-life allows high-throughput batchprocessing.Signal stability is shown for three common cell lines. HepG2 and BHK-21cells were grown and assayed in MEM containing 10% FBS, while CHO-K1 cells were grown and assayed in DME/F-12 containing 10% FBS. CHO-K1, BHK-21 and HepG2 cells, at 25,000 cells per well, were added to a 96-well plate. After an equal volume of CellTiter-Glo ®Reagent was added, plates were shaken and luminescence monitored over time with the plates held at 22°C. The half-lives of the luminescent signals for the CHO-K1, BHK-21 and HepG2 cells were approximately 5.4, 5.2 and5.8hours, respectively.2.Product Components and Storage ConditionsProduct Size Cat.#CellTiter-Glo ®Luminescent Cell Viability Assay 10ml G7570Substrate is sufficient for 100 assays at 100µl/assay in 96-well plates or 400 assays at 25µl/assay in 384-well plates. Includes:• 1 × 10mlCellTiter-Glo ®Buffer • 1 vial CellTiter-Glo ®Substrate (lyophilized)Product Size Cat.#CellTiter-Glo ®Luminescent Cell Viability Assay 10 × 10ml G7571Each vial of substrate is sufficient for 100 assays at 100µl/assay in 96-well plates or 400 assays at 25µl/assay in 384-well plates (1,000 to 4,000 total assays). Includes:•10 × 10mlCellTiter-Glo ®Buffer •10 vials CellTiter-Glo ®Substrate (lyophilized)Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·R e l a t i v e L u m i n e s c e n c e (%)Time (minutes)CHO-K101020304050607080901003173M A 12_0AProduct Size Cat.# CellTiter-Glo®Luminescent Cell Viability Assay100ml G7572 Substrate is sufficient for 1,000 assays at 100µl/assay in 96-well plates or 4,000assays at 25µl/assay in 384-well plates. Includes:•1 × 100ml CellTiter-Glo®Buffer• 1 vial CellTiter-Glo®Substrate (lyophilized)Product Size Cat.# CellTiter-Glo®Luminescent Cell Viability Assay10 × 100ml G7573Each vial of substrate is sufficient for 1,000 assays at 100µl/assay in 96-well plates or4,000 assays at 25µl/assay in 384-well plates (10,000to 40,000 total assays). Includes:•10 × 100ml CellTiter-Glo®Buffer•10 vials CellTiter-Glo®Substrate (lyophilized)Storage Conditions:For long-term storage, store the lyophilized CellTiter-Glo®Substrate and CellTiter-Glo®Buffer at –20°C. For frequent use, the CellTiter-Glo®Buffer can be stored at 4°C or room temperature for 48hours without loss of activity. See product label for expiration date information. ReconstitutedCellTiter-Glo®Reagent (Buffer plus Substrate) can be stored at room temperaturefor up to 8hours with <10% loss of activity, at 4°C for 48hours with ~5% lossof activity, at 4°C for 4days with ~20% loss of activity or at –20°C for 21weekswith ~3% loss of activity. The reagent is stable for up to ten freeze-thaw cycles,with less than 10% loss of activity.3.Performing the CellTiter-Glo®AssayMaterials to Be Supplied by the User•opaque-walled multiwell plates adequate for cell culture•multichannel pipette or automated pipetting station for reagent delivery•device (plate shaker) for mixing multiwell plates•luminometer, CCD camera or imaging device capable of reading multiwell plates •optional:ATP for use in generating a standard curve (Section 3.C)3.A.Reagent Preparation1.Thaw the CellTiter-Glo®Buffer, and equilibrate to room temperature priorto use. For convenience the CellTiter-Glo®Buffer may be thawed andstored at room temperature for up to 48hours prior to use.2.Equilibrate the lyophilized CellTiter-Glo®Substrate to room temperatureprior to use.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3.A.Reagent Preparation (continued)3.Transfer the appropriate volume (10ml for Cat.# G7570 and G7571, or 100mlfor Cat.# G7572 and G7573) of CellTiter-Glo ®Buffer into the amber bottlecontaining CellTiter-Glo ®Substrate to reconstitute the lyophilizedenzyme/substrate mixture. This forms the CellTiter-Glo ®Reagent.4.Mix by gently vortexing, swirling or inverting the contents to obtain ahomogeneous solution. The CellTiter-Glo ®Substrate should go intosolution easily in less than 1minute.3.B.Protocol for the Cell Viability AssayWe recommend that you perform a titration of your particular cells todetermine the optimal number and ensure that you are working within thelinear range of the CellTiter-Glo ®Assay. Figure 2 provides an example of sucha titration of HEK293 cells using 0 to 50,000 cells per well in a 96-well format.1.Prepare opaque-walled multiwell plates with mammalian cells in culturemedium, 100µl per well for 96-well plates or 25µl per well for 384-wellplates.Multiwell plates must be compatible with the luminometer used.2.Prepare control wells containing medium without cells to obtain a value forbackground luminescence.3.Add the test compound to experimental wells, and incubate according toculture protocol.4.Equilibrate the plate and its contents at room temperature forapproximately 30 minutes.5.Add a volume of CellTiter-Glo ®Reagent equal to the volume of cell culturemedium present in each well (e.g., add 100µl of reagent to 100µl of mediumcontaining cells for a 96-well plate, or add 25µl of reagent to 25µl ofmedium containing cells for a 384-well plate).6.Mix contents for 2 minutes on an orbital shaker to induce cell lysis.7.Allow the plate to incubate at room temperature for 10 minutes to stabilizeluminescent signal.Note:Uneven luminescent signal within standard plates can be caused bytemperature gradients, uneven seeding of cells or edge effects in multiwellplates.8.Record luminescence.Note:Instrument settings depend on the manufacturer. An integration timeof 0.25–1 second per well should serve as a guideline.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·3.C.Protocol for Generating an ATP Standard Curve (optional)It is a good practice to generate a standard curve using the same plate onwhich samples are assayed. We recommend ATP disodium salt (Cat.# P1132,Sigma Cat.# A7699 or GE Healthcare Cat.# 27-1006). The ATP standard curveshould be generated immediately prior to adding the CellTiter-Glo®Reagentbecause endogenous ATPase enzymes found in sera may reduce ATP levels.1.Prepare 1µM ATP in culture medium (100µl of 1µM ATP solution contains10–10moles ATP).2.Prepare serial tenfold dilutions of ATP in culture medium (1µM to 10nM;100µl contains 10–10to 10–12moles of ATP).3.Prepare a multiwell plate with varying concentrations of ATP standard in100µl medium (25µl for a 384-well plate).4.Add a volume of CellTiter-Glo®Reagent equal to the volume of ATPstandard present in each well.5.Mix contents for 2 minutes on an orbital shaker.6.Allow the plate to incubate at room temperature for 10 minutes to stabilizethe luminescent signal.7.Record luminescence.4.Appendix4.A.Overview of the CellTiter-Glo®AssayThe assay system uses the properties of a proprietary thermostable luciferase toenable reaction conditions that generate a stable “glow-type” luminescentsignal while simultaneously inhibiting endogenous enzymes released duringcell lysis (e.g., ATPases). Release of ATPases will interfere with accurate ATPmeasurement. Historically, firefly luciferase purified from Photinus pyralis(LucPpy) has been used in reagents for ATP assays (1,4–7). However, it hasonly moderate stability in vitro and is sensitive to its chemical environment,including factors such as pH and detergents, limiting its usefulness fordeveloping a robust homogeneous ATP assay. Promega has successfullydeveloped a stable form of luciferase based on the gene from another firefly,Photuris pennsylvanica(LucPpe2), using an approach to select characteristics thatimprove performance in ATP assays. The unique characteristics of this mutant(LucPpe2m) enabled design of a homogeneous single-reagent-addition approachto perform ATP assays with cultured cells. Properties of the CellTiter-Glo®Reagent overcome the problems caused by factors, such as ATPases, thatinterfere with ATP measurement in cell extracts. The reagent is physicallyrobust and provides a sensitive and stable luminescent output.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·4.A.Overview of the CellTiter-Glo®Assay (continued)Sensitivity and Linearity:The ATP-based detection of cells is more sensitivethan other methods (8–10). In experiments performed by Promega scientists,the luminescent signal from 50HEK293 cells is greater than three standarddeviations above the background signal from serum-supplemented mediumwithout cells. There is a linear relationship (r2= 0.99) between the luminescentsignal and the number of cells from 0 to 50,000 cells per well in the 96-wellformat. The luminescence values in Figure 2 were recorded after 10minutes ofincubation at room temperature to stabilize the luminescent signal as describedin Section3.B. Incubation of the same 96-well plate used in the experimentshown in Figure 2 for 360minutes at room temperature had little effect on therelationship between luminescent signal and number of cells (r2= 0.99).Speed:The homogeneous procedure to measure ATP using the CellTiter-Glo®Assay is quicker than other ATP assay methods that require multiple steps toextract ATP and measure luminescence. The CellTiter-Glo®Assay also is fasterthan other commonly used methods to measure the number of viable cells(such as MTT, alamarBlue®or Calcein-AM) that require prolonged incubationsteps to enable the cells’ metabolic machinery to convert indicator moleculesinto a detectable signal.4.B.Additional ConsiderationsTemperature:The intensity and decay rate of the luminescent signal from theCellTiter-Glo®Assay depends on the luciferase reaction rate. Environmentalfactors that affect the luciferase reaction rate will change the intensity andstability of the luminescent signal. Temperature is one factor that affects therate of this enzymatic assay and thus the light output. For consistent results,equilibrate assay plates to a constant temperature before performing the assay.Transferring eukaryotic cells from 37°C to room temperature has little effect onATP content (5). We have demonstrated that removing cultured cells from a37°C incubator and allowing them to equilibrate to 22°C for 1–2 hours hadlittle effect on ATP content. For batch-mode processing of multiple assayplates, take precautions to ensure complete temperature equilibration. Platesremoved from a 37°C incubator and placed in tall stacks at room temperaturewill require longer equilibration than plates arranged in a single layer.Insufficient equilibration may result in a temperature gradient effect betweenwells in the center and at the edge of the plates. The temperature gradientpattern also may depend on the position of the plate in the stack.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·Chemicals:The chemical environment of the luciferase reaction affects theenzymatic rate and thus luminescence intensity. Differences in luminescenceintensity have been observed using different types of culture media and sera.The presence of phenol red in culture medium should have little impact onluminescence output. Assaying 0.1µM ATP in RPMI medium without phenolred resulted in ~5% increase in luminescence output (in relative light units[RLU]) compared to assays in RPMI containing the standard concentration ofphenol red, whereas assays in RPMI medium containing twice the normalconcentration of phenol red showed a ~2% decrease in luminescence.Solvents for the various test compounds may interfere with the luciferasereaction and thus the light output from the assay. Interference with theluciferase reaction can be detected by assaying a parallel set of control wellscontaining medium without cells. Dimethylsulfoxide (DMSO), commonly usedas a vehicle to solubilize organic chemicals, has been tested at finalconcentrations of up to 2% in the assay and only minimally affects light output.Plate Recommendations:We recommend using standard opaque-walledmultiwell plates suitable for luminescence measurements. Opaque-walledplates with clear bottoms to allow microscopic visualization of cells also maybe used; however, these plates will have diminished signal intensity andgreater cross talk between wells. Opaque white tape may be used to decreaseluminescence loss and cross talk.Cellular ATP Content:Different cell types have different amounts of ATP,and values reported for the ATP level in cells vary considerably (1,4,11–13).Factors that affect the ATP content of cells may affect the relationship betweencell number and luminescence. Anchorage-dependent cells that undergocontact inhibition at high densities may show a change in ATP content per cellat high densities, resulting in a nonlinear relationship between cell numberand luminescence. Factors that affect the cytoplasmic volume or physiology ofcells also will affect ATP content. For example, oxygen depletion is one factorknown to cause a rapid decrease in ATP (1).Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll F ree in USA 800-356-9526·Phone 608-274-4330 ·F ax 608-277-2516 ·4.B.Additional Considerations (continued)Mixing:Optimal assay performance is achieved when the CellTiter-Glo®Reagent is mixed completely with the cultured cells. Suspension cell lines (e.g., Jurkat cells) generally require less mixing to achieve lysis and extract ATP than adherent cells (e.g., L929 cells). Tests were done to evaluate the effect ofshaking the plate after adding the CellTiter-Glo® Reagent. Suspension cellscultured in multiwell plates showed only minor differences in light outputwhether or not the plates were shaken after adding the CellTiter-Glo®Reagent.Adherent cells are more difficult to lyse and show a substantial differencebetween shaken and nonshaken plates.Several additional parameters related to reagent mixing include the force ofdelivery of CellTiter-Glo®Reagent, sample volume and dimensions of the well.All of these factors may affect assay performance. The degree of reagent mixing required may be affected by the method used to add the CellTiter-Glo®Reagent to the assay plates. Automated pipetting devices using a greater or lesser force of fluid delivery may affect the degree of subsequent mixing required.Complete reagent mixing in 96-well plates should be achieved using orbitalplate shaking devices built into many luminometers and the recommended2-minute shaking time. Special electromagnetic shaking devices that use aradius smaller than the well diameter may be required to efficiently mixcontents of 384-well plates. The depth of medium and geometry of themultiwell plates may have an effect on mixing efficiency. We recommend that you take these factors into consideration when performing the assay andempirically determine whether a mixing step is necessary for the individualapplication.LuminometersFor highly sensitive luminometric assays, the luminometer model and settings greatly affect the quality of data obtained. Luminometers from differentmanufacturers will vary in sensitivities and dynamic ranges. We recommend the GloMax®products because these instruments do not require gainadjustments to achieve optimal sensitivity and dynamic range. Additionally, GloMax®instruments are preloaded with Promega protocols for ease of use.If you are not using a GloMax®luminometer, consult the operating manual for your luminometer to determine the optimal settings. The limits should beverified on each instrument before analysis of experimental samples. The assay should be linear in some portion of the detection range of the instrument used.For an individual luminometer there may be different gain settings. Werecommend that you optimize the gain settings.4.C.References1.Crouch, S.P. et al.(1993) The use of ATP bioluminescence as a measure of cellproliferation and cytotoxicity. J. Immunol. Methods160, 81–8.2.Farfan, A.et al.(2004) Multiplexing homogeneous cell-based assays. Cell Notes10, 2–5.3.Riss, T., Moravec, R. and Niles, A. (2005) Selecting cell-based assays for drugdiscovery screening. Cell Notes13, 16–21.4.Kangas, L., Grönroos, M. and Nieminen, A.L. (1984) Bioluminescence of cellular ATP:A new method for evaluating cytotoxic agents in vitro. Med. Biol.62, 338–43.5.Lundin, A. et al.(1986) Estimation of biomass in growing cell lines by adenosinetriphosphate assay.Methods Enzymol. 133, 27–42.6.Sevin, B.U. et al.(1988) Application of an ATP-bioluminescence assay in human tumorchemosensitivity testing. Gynecol. Oncol.31, 191–204.7.Gerhardt, R.T.et al.(1991) Characterization of in vitro chemosensitivity ofperioperative human ovarian malignancies by adenosine triphosphatechemosensitivity assay. Am. J. Obstet. Gynecol. 165, 245–55.8.Petty, R.D. et al.(1995) Comparison of MTT and ATP-based assays for themeasurement of viable cell number. J. Biolumin. Chemilumin.10, 29–34.9.Cree, I.A. et al.(1995) Methotrexate chemosensitivity by ATP luminescence in humanleukemia cell lines and in breast cancer primary cultures: Comparison of the TCA-100assay with a clonogenic assay. AntiCancer Drugs6, 398–404.10.Maehara, Y. et al.(1987) The ATP assay is more sensitive than the succinatedehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer Clin. Oncol.23, 273–6.11.Stanley, P.E. (1986) Extraction of adenosine triphosphate from microbial and somaticcells. Methods Enzymol.133, 14–22.12.Beckers, B. et al.(1986) Application of intracellular ATP determination in lymphocytesfor HLA-typing. J. Biolumin. Chemilumin.1, 47–51.13.Andreotti, P.E. et al.(1995) Chemosensitivity testing of human tumors using amicroplate adenosine triphosphate luminescence assay: Clinical correlation forcisplatin resistance of ovarian carcinoma. Cancer Res. 55, 5276–82.4.D.Related ProductsCell Proliferation ProductsProduct Size Cat.# ApoLive-Glo™ Multiplex Assay10ml G6410 ApoTox-Glo™ Triplex Assay10ml G6320 CellTiter-Fluor™ Cell Viability Assay (fluorescent)10ml G6080 CellTiter-Blue®Cell Viability Assay (resazurin)20ml G8080 CellTiter 96®AQ ueous One SolutionCell Proliferation Assay (MTS, colorimetric)200 assays G3582 CellTiter 96®AQ ueous Non-RadioactiveCell Proliferation Assay (MTS, colorimetric)1,000 assays G5421 CellTiter 96®AQ ueous MTS Reagent Powder1g G1111 CellTiter 96®Non-RadioactiveCell Proliferation Assay (MTT, colorimetric)1,000 assays G4000 Additional sizes available.Cytotoxicity AssaysProduct Size Cat.# CytoTox-Glo™ Cytotoxicity Assay (luminescent)*10ml G9290Mitochondrial ToxGlo™ Assay*10ml G8000 MultiTox-Glo Multiplex Cytotoxicity Assay(luminescent, fluorescent)*10ml G9270 MultiTox-Fluor Multiplex Cytotoxicity Assay(fluorescent)*10ml G9200 CytoTox-Fluor™ Cytotoxicity Assay (fluorescent)*10ml G9260 CytoTox-ONE™ Homogeneous MembraneIntegrity Assay (LDH, fluorometric)*200–800 assays G7890 CytoTox-ONE™ Homogeneous MembraneIntegrity Assay, HTP1,000–4,000 assays G7892 CytoTox 96® Non-Radioactive Cytotoxicity Assay1,000 assays G1780 (LDH, colorimetric)*GSH-Glo™ Glutathione Assay10ml V691150ml V6912 GSH/GSSG-Glo™ Assay10ml V661150ml V6612 *Additional sizes available.LuminometersProduct Size Cat.# GloMax®-Multi+ Detection System with Instinct™ Software:Base Instrument with Shaking 1 each E8032 GloMax®-Multi+ Detection System with Instinct™ Software:Base Instrument with Heating and Shaking 1 each E9032 GloMax®-Multi+ Luminescence Module 1 each E8041Apoptosis ProductsProduct Size Cat.# Caspase-Glo®2 Assay*10ml G0940 Caspase-Glo®6 Assay*10ml G0970 Caspase-Glo®3/7 Assay* 2.5ml G8090 Caspase-Glo®8 Assay* 2.5ml G8200 Caspase-Glo®9 Assay* 2.5ml G8210Apo-ONE®Homogeneous Caspase-3/7 Assay1ml G7792 DeadEnd™ Fluorometric TUNEL System60 reactions G3250 DeadEnd™ Colorimetric TUNEL System20 reactions G7360Anti-ACTIVE®Caspase-3 pAb50µl G7481Anti-PARP p85 Fragment pAb50µl G7341Anti-pS473Akt pAb40µl G7441 Caspase Inhibitor Z-VAD-FMK, 20mM50µl G7231125µl G7232*Additional sizes available.(a)U.S. Pat. Nos. 6,602,677 and 7,241,584, European Pat. No. 1131441, Japanese Pat. Nos. 4537573 and 4520084 and other patents pending(b)U.S. Pat. No. 7,741,067, Japanese Pat. No. 4485470 and other patents pending.(c)U.S. Pat. No. 7,700,310, European Pat. No. 1546374 and other patents pending.(d)U.S. Pat. Nos 7,083,911, 7,452,663 and 7,732,128, European Pat. No. 1383914 and Japanese Pat. Nos. 4125600 and 4275715.(e)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.© 2001–2012 Promega Corporation. All Rights Reserved.Anti-ACTIVE, Apo-ONE, Caspase-Glo, CellTiter 96, CellTiter-Blue, CellTiter-Glo, CytoTox 96 and GloMax are registered trademarks of Promega Corporation. ApoTox-Glo, ApoLive-Glo, CellTiter-Fluor, CytoTox-Fluor, CytoTox-Glo, CytoTox-ONE, DeadEnd, GSH-Glo, GSH/GSSG-Glo, Instinct, Mitochondrial ToxGlo and Ultra-Glo are trademarks of Promega Corporation. alamarBlue is a registered trademark of Trek Diagnostic Ssystems, Inc.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.。

Cell-BasedAssay（细胞活性、毒性和凋亡）

•定量计算某种药物或某荧光法: 刃天青
种因素对细胞的毒性或促增殖的作用
生物发光法：ATP法
•作为其他实验的对照
同位素标记：H3Thymidine标记
高灵敏度，高通量，操作简单
高灵敏度，高通量，操作时间短
灵敏度高，但是操作复杂，放射性方法，成本高
MTT操作步骤
前期培养
加加DDyyeeSSooluluttioionn
Vijaya Ramachandran, Thiruvengadam Arumugam, Huamin Wang, and Craig D. Logsdon Cancer Res., Oct 2008; 68: 7811 - 7818.
5.NADPH Oxidase-dependent Signaling Involved In Endothelial Cell Survival And Proliferation: Role Of Nox2 And Nox4
出？ • 需要叠加其他实验？
如何选择适合的检测方法
• 现有的仪器 • 通量和灵敏度 • 操作简便度和操作时间的要求
对实验效率的要求-高通量药物筛选
实验目的与常用方法
实验目的
方法
•初步观察细胞的生存状态，镜下计算活细胞和死台盼蓝染料排斥法细胞比例
特点
镜下观察计数细胞
比色法：MTT,MTS 中低通量，操作简单
Lan H. Ly, Xiao-Yan Zhao, Leah Holloway, and David Feldman.Endocrinology, May 1999; 140: 2071.
4.Anterior Gradient 2 Is Expressed and Secreted during the Development of Pancreatic Cancer and Promotes Cancer Cell Survival

Caspase-3活性荧光检测试剂盒

Caspase-3舌性荧光检测试剂盒Caspase （天胱蛋白酶）家族在介导细胞凋亡的过程中起着非常重要的作用，其中Caspase-3是凋亡信号通路中一个重要的蛋白酶，也是凋亡的关键执行分子。

Caspase-3正常以酶原（32KD）的形式存在于胞浆中，没有活性。

在凋亡的早期阶段，它被激活；活化的Caspase-3由两个大亚基（17KD）和两个小亚基（12KD）组成，裂解相应的胞浆、胞核底物，最终导致细胞凋亡。

但处于晚期凋亡以及死亡的细胞中Caspase-3的活性明显下降，会影响Caspase3活性检测结果，建议选择早期和中期凋亡的细胞进行检测。

一原理：在凋亡早期阶段，活化的Caspase-3能够特异地剪切D1E2V3D4-X底物，水解D4-X肽键。

根据这一特点，设计出荧光物质偶联的短肽Ac-DEVD-AMC在共价偶联时，发光基团AMC不能被激发荧光，短肽Ac-DEVD-AMC被水解后释放出发光基团AMC，游离的AMC才能被激发发射荧光。

根据释放的AMC荧光强度的大小，可以测定Caspase-3的活性，从而反映Caspase-3被活化的程度。

二方法：（一）细胞总蛋白提取和定量：选用适当方法诱导细胞凋亡，同时设阴性对照组。

以下操作均在冰上或4°C 进行，所有液体需事先预冷：仁收获正常和凋亡细胞，用PBS洗细胞2次，将其转入EP管，离心并弃尽上清；2. 将细胞重悬于加入Cocktail的细胞裂解液中，1X106细胞约加50 l裂解液（视细胞多少而定加入裂解液的量），混匀后冰浴30分钟；（此步可加入细胞裂解液后超声裂解细胞）。

3•离心12000rpm/10分钟，收集上清，即为细胞总蛋白；4•取少量上清（1 —2"），用Bradford或BCA法进行蛋白质定量，检测前将蛋白用细胞裂解液（含Cocktai）稀释为2 g/ I。

（二）体外Caspase-3的活性检测：1. 实验设空白对照孔和待测样品孔，均加入含荧光底物Ac-DEVD-AMC勺Caspase-3反应缓冲液（反应缓冲液使用前加入Reagent1和2）45 l/孔；2•空白孔加5 1/孔正常细胞裂解液（含Cocktail）,待测样品孔加10 g/孔（5 l）细胞总蛋白，总量50 l/孔。

kinase-glo

kinase-gloKinase-Glo是一种广泛应用于生物学研究中的试剂盒，用于测定激酶活性。

下面我将从多个角度进行全面回答。

1. 什么是Kinase-Glo？Kinase-Glo是一种商业化的试剂盒，由Promega公司开发和销售。

它是一种用于测定激酶活性的试剂盒，可以测量细胞内或体外激酶的磷酸化活性。

2. Kinase-Glo的工作原理是什么？Kinase-Glo试剂盒的工作原理基于荧光检测技术。

首先，该试剂盒中的底物被激酶磷酸化，生成磷酸化产物。

然后，加入试剂盒中的检测底物会与磷酸化产物发生反应，释放出荧光信号。

最后，使用荧光酶标仪或荧光显微镜等设备检测并测量荧光信号的强度，从而确定激酶的活性。

3. Kinase-Glo适用于哪些实验研究？Kinase-Glo广泛应用于各种生物学研究领域，特别是与蛋白激酶相关的研究。

它可以用于测定细胞内或体外的激酶活性，研究激酶的底物特异性、受体激酶的激活、抑制剂的筛选等。

4. 使用Kinase-Glo有哪些优势？Kinase-Glo具有以下几个优势：灵敏度高，Kinase-Glo可以检测低浓度的激酶活性，具有较高的灵敏度。

高通量，该试剂盒适用于高通量筛选，可以同时测定多个样品的激酶活性。

快速，Kinase-Glo的操作简便，整个实验过程快速，可以在短时间内获得结果。

可靠性，该试剂盒具有较好的重复性和稳定性，可以得到可靠的实验结果。

5. 如何正确使用Kinase-Glo试剂盒？使用Kinase-Glo试剂盒时，需要按照产品说明书中的操作步骤进行。

一般包括以下几个步骤，准备样品和底物，将样品和底物混合，孵育一段时间以使激酶反应发生，加入检测底物，测量荧光信号强度。

同时，注意使用质量控制和标准曲线来确保实验结果的准确性和可靠性。

总结起来，Kinase-Glo是一种用于测定激酶活性的试剂盒，通过荧光检测技术测量激酶反应产生的磷酸化产物，并得出激酶的活性结果。

它在生物学研究中具有广泛的应用，可用于研究蛋白激酶的底物特异性、受体激酶的激活、抑制剂的筛选等。

caspase-glo3 7assay

1.说明2.G8090成分1x2.5ml caspase 3/7 buffer，1瓶caspase 3/7底物（冻干）；置于-20度，避光。

4度3个月，常温4天。

96孔板25次100ul/孔384孔板100次25 ul/孔3.试剂的准备和储存a caspase 3/7 buffer和caspase 3/7底物用前置于室温；b 将caspase 3/7 buffer加入caspase 3/7底物瓶，混匀，使得caspase 3/7底物溶解，成为caspase glo-3/7试剂。

c caspase glo-3/7试剂4度可放置3天；4度放置1周可达到90%信号；4度放置4周可达到75%信号；caspase glo-3/7试剂-20度放置1周可达到75%信号；-20度放置4周可达到60%信号。

4. 检测细胞内caspase glo-3/7活性本实验caspase glo-3/7试剂和样品1：1混合。

（96孔板100ul caspase glo-3/7试剂+100ul 样品；384孔板25ul caspase glo-3/7试剂+100ul样品。

）需要准备的仪器a 光度计b 96孔板振荡器c 加样器d 96孔板4 A检测条件空白对照：caspase glo-3/7试剂+细胞培养液阴性对照：caspase glo-3/7试剂+培养细胞的细胞培养液实验孔：caspase glo-3/7试剂+处理后细胞的细胞培养液注意：a 96孔板中加入<20000细胞/孔；b 阴性对照和试验孔细胞数要一致；c 最高发光信号可能在1-2h达到，所以要在3h之内检测。

4 B 96孔板中培养的细胞caspase glo-3/7活性检测的标准方案a 检测前，把caspase 3/7 buffer和caspase 3/7底物置于室温，混匀；b 拿出96孔板，室温放置片刻；c 把caspase glo-3/7试剂100ul加入96孔板中（空白孔，阴性对照孔，试验孔）。

藏红花素对耐伊马替尼慢性白血病粒细胞的促凋亡作用

藏红花素对耐伊马替尼慢性白血病粒细胞的促凋亡作用张洋;郑卫东;邸玉玮;方伟;廖玉琴【摘要】[Objective] To investigate the proliferative inhibition and apoptotic induction effect of crocin on imatinib-resistant K562/Ima cells.[Methods] K562/Ima cells were seeded in the 96-well plant and treated with different concentrations of crocin for 48 h.Then CCK8 method was applied to explore the inhibition rate of crocin.The apoptosis rate of crocin on K562/Ima cells was detected by Hoechst 33258 dying and Annexin V/PI method.Caspase activity was detected after crocin treatment.[Results] Crocin could effectively inhibit K562/Ima cells growth,with a concentration dependent pattern.The apoptotic rate ofK562/Ima cells was increased as the concentration of crocin wasraised.Caspase-3 was activated after crocin treatment in K562/Imacells.Caspase inhibitor Z-VAD-FMK could effectively revise thiseffect.[Conclusion] Crocin can significantly inhibit growth and induce apoptosis of K562/Ima cells probably via Caspase pathway.%[目的]分析探讨藏红花素对耐伊马替尼白血病细胞株K562/Ima的增殖抑制和促凋亡的机制.[方法]耐伊马替尼的白血病细胞株K562/Ima接种于96孔板中,经不同浓度的藏红花素分别处理48h采用CCK8法检测细胞增殖抑制情况,Hoechst染色藏红花素处理的细胞,荧光显微镜观察及采用Annexin V/PI双染分析藏红花素对K562/Ima细胞的促凋亡作用.K562/Ima细胞经藏红花素处理,及采用Caspase蛋白抑制剂处理,检测Caspase-3激活情况.[结果]细胞增殖抑制实验显示藏红花素能够显著抑制耐药K562/Ima细胞的增殖,呈一定的浓度依赖关系,P＜0.05.随着藏红花素浓度的提高,K562/Ima细胞凋亡率逐渐上升,表现为细胞皱缩,细胞核固缩,染色增强,出现核小体.荧光活性检测实验显示经藏红花素处理后,Caspase-3活性明显增强.采用Caspase抑制剂Z-VAD-FMK作用后,Caspase-3活性被部分抑制,K562/Ima细胞凋亡率降低.[结论]藏红花素能够抑制K562/Ima细胞的增殖,诱导其凋亡,Caspase 信号通路可能参与其中.【期刊名称】《中山大学学报（医学科学版）》【年(卷),期】2013(034)004【总页数】5页(P537-541)【关键词】藏红花素;伊马替尼;慢性粒细胞性白血病;细胞凋亡【作者】张洋;郑卫东;邸玉玮;方伟;廖玉琴【作者单位】广东省医学科学院//广东省人民医院检验科,广东广州510080;广东省医学科学院//广东省人民医院检验科,广东广州510080;广东省医学科学院//广东省人民医院检验科,广东广州510080;广东省医学科学院//广东省人民医院检验科,广东广州510080;广东省医学科学院//广东省人民医院检验科,广东广州510080【正文语种】中文【中图分类】R73-36慢性粒细胞白血病（chronic myeloid leukemia，CML），是一种起源于造血干细胞的恶性增殖性疾病［1-2］，病程通常分为慢性期、加速期和急变期［3］。

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Revised 12/12TB323T E C H N I C A L B U L L E T I NCaspase-Glo® 3/7 AssayInstrucƟ ons for Use of ProductsG8090, G8091, G8092 and G8093Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1 TB323 · Revised 12/12All technical literature is available at: /protocols/Visit the web site to verify that you are using the most current version of this Technical Bulletin. E-mail Promega Technical Services if you have questions on use of this system: techserv@Caspase-Glo ® 3/7 Assay1. Desc ription .........................................................................................................................................1 2. Product Components and Storage Conditions ........................................................................................4 3. Reagent Preparation and Storage .........................................................................................................6 4. Detection of Caspase-3 and -7 Activities in Cell-Based Assays .. (6)4.A. Assay Conditions ........................................................................................................................64.B. Standard Protocol for Cells Cultured in a 96-Well Plate .................................................................9 5. Detection of Caspase-3 or -7 Activity Using Puriﬁ ed Caspases .. (9)5.A. Assay Conditions ........................................................................................................................95.B. Standard Protocol for Puriﬁ ed Caspase Enzyme ..........................................................................106. General Considerations .....................................................................................................................147. Referenc es ........................................................................................................................................168. Related Produc ts ...............................................................................................................................171. DescriptionThe Caspase-Glo ® 3/7 Assay (a–c) is a homogeneous, luminescent assay that measures caspase-3 and -7 activities. These members of the cysteine aspartic acid-speciﬁ c protease (caspase) family play key eﬀ ector roles in apoptosis in mammalian cells (1–7). The assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Adding a singleCaspase-Glo ® 3/7 Reagent in an “add-mix-measure” format results in cell lysis, followed by caspase cleavage of the substrate and generation of a “glow-type” luminescent signal, produced by luciferase (Figure 1). Luminescence is proportional to the amount of caspase activity present (Figure 2). The Caspase-Glo ® 3/7 Reagent relies on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase), which is formulated to generate a stable “glow-type” luminescent signal and improve performance across a wide range of assay conditions.The Caspase-Glo ® 3/7 Assay is designed for use with multiwell-plate formats, making it ideal for automated high-throughput screening of caspase activity or apoptosis. The assay has been automated in 96-, 384- and 1536-well formats. Cell washing, removing medium and multiple pipetting steps are not required (Figure 3). The caspase and luciferase enzyme activities reach steady state so that the luminescent signal peaks in approximately one hour and is maintained for several hours with a minimal loss of signal (Figures 5 and 6). This results in a rapid, sensitive and ﬂ exible caspase-3/7 activity assay. The assay system may be used with puriﬁ ed enzyme preparations (Figure 2) or cultures of adherent or suspension cells (Figure 5). We have tested this assay on a variety of cell lines (Jurkat, L929,2Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516TB323 · Revised 12/121. Description (continued)HeLa, HL-60, SH-SY5Y, HepG2) that have been exposed to several diﬀ erent drugs and apoptosis-inducing agents (anti-Fas, TNF-α, staurosporine, clozapine, vinblastine, tamoxifen). This assay also can be multiplexed with other homogeneous assays to measure more than one parameter from a single well (Figure 8).4061M B 04_3ANS HSN Z-DEVD-NCOOH-NS SN H 2NCOOH+ ATP + O 2Z-DEVD- +Figure 1. Caspase-3/7 cleavage of the luminogenic substrate containing the DEVD sequence. Following caspase cleavage, a substrate for luciferase (aminoluciferin) is released, resulting in the luciferase reaction and the production of light.Advantages of the Caspase-Glo ® 3/7 Assay include:Simple Method: The homogeneous “add-mix-read” protocol makes the assay amenable to automation in 96-, 384- or 1536-well formats (Figures 3 and 8). For information about obtaining automated protocols, see: /automethodsGreater Sensitivity: The Caspase-Glo ® 3/7 Assay is more sensitive than ﬂ uorescence-based caspase assays(Figure 4; 8–10). Maximum sensitivity is typically reached within 1 hour. The luminescent assay avoids interference from ﬂ uorescence signals of test compounds, providing excellent signal-to-noise ratios. The sensitivity allows you to use fewer cells if monitoring apoptosis, or less puriﬁ ed caspase if screening for caspase inhibitors.Fast: No sample preparation or manipulation is required. The Caspase-Glo ® 3/7 Assay does not require extended incubation times to accumulate product to reach maximum sensitivity, in contrast to ﬂ uorescence-based assays.Robust: The Caspase-Glo ® 3/7 Assay delivers excellent Z´-factor values in cell and puriﬁ ed enzyme models (see Figure 7). Z´-factor values greater than 0.5 indicate excellent assay quality (11).Amenable to Batch Processing: The extended glow signal allows plates to be read over an extended period of time (Figures 5 and 6); there is no need to use luminometers with reagent injectors.Selective for Caspase-3 and -7: The Caspase-Glo ® 3/7 Assay uses a luminogenic substrate containing the DEVD sequence, which has been shown to be selective for caspase-3 and -7 (5).Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 3 TB323 · Revised 12/12Flexible: The simpliﬁ ed format involves adding an equal volume of reagent to the sample, allowing one to thousands of assays to be performed.Amenable to Multiplexing: The Caspase-Glo ® 3/7 Assay can be multiplexed with other homogeneous assays to measure multiple parameters from a single sample (12,13).4097M A 04_3ACaspase-3 (Uni t s/Well)L u m i n e s c e n c e (R L U , B l a n k S u b t r a c t e d )Figure 2. Luminescence is proportional to caspase-3 activity. Puriﬁ ed caspase-3 was titrated and assayed in a total volume of 200µl per well in a 96-well plate. Luminometer readings were taken 1 hour after adding the Caspase-Glo ® 3/7 Reagent. The assay is linear over 4 orders of magnitude of caspase concentration (R 2 = 0.998, slope = 0.989)1. One unit caspase (0.07ng protein) is the amount of enzyme required to cleave 1pmol of substrate (Ac-DEVD-pNA) hydrolyzed/minute at 30°C per the manufacturer’s unit deﬁ nition 2. Each point represents the average of 4 wells. Values are blank-subtracted (blank = no caspase).1Determined by converting the log graph to a log 10 linear plot to calculate r 2 and slope.2Unit deﬁ nitions of caspase-3 activity may vary between manufacturers, so the number of units may not translate directly between vendors. Notes: 1) Due to the extended dynamic range of of the Caspase-Glo ® 3/7 Assay, data were graphed on a log scale. 2) The number of relative light units (RLU) obtained will depend on the type of luminometer used. For this reason, and because unit deﬁ nitions may vary, your results may diﬀ er.Selected Citations Using the Caspase-Glo ® 3/7 Assay:• Liu, D. et al. (2004) Nuclear import of proinﬂ ammatory transcription factors is required for massive liverapoptosis induced by bacterial lipopolysaccharide. J. Biol. Chem. 279, 48434–42. The Caspase-Glo ® 3/7, -8 and -9 Assays were used to measure caspase-3, -8, and -9 activities in mouse liverhomogenates.• Ren, Y.G. et al. (2004) Diﬀ erential regulation of the TRAIL death receptors DR4 and DR5 by the signalrecognition particle. Mol. Biol. Cell 15, 5064–74.Caspase activation was measured using the Caspase-Glo ® 3/7 Reagent in HCT15 cells.For additional peer-reviewed articles that cite the Caspase-Glo ® 3/7 Assay, please visit: /citations/2. Product Components and Storage ConditionsP R O D U C T S I Z E C AT.# Caspase-Glo® 3/7 Assay 2.5ml G8090 Each system contains suﬃ cient reagents for 25 assays at 100µl per assay in a 96-well plate or 100 assays of25µl per assay in a 384-well plate. Includes:• 1 × 2.5ml Caspase-Glo® 3/7 Buﬀ er• 1 bottle Caspase-Glo® 3/7 Substrate (lyophilized)P R O D U C T S I Z E C AT.# Caspase-Glo® 3/7 Assay 10ml G8091 Each system contains suﬃ cient reagents for 100 assays at 100µl per assay in a 96-well plate or 400 assays of 25µl per assay in a 384-well plate. Includes:• 1 × 10ml Caspase-Glo® 3/7 Buﬀ er• 1 bottle Caspase-Glo® 3/7 Substrate (lyophilized)P R O D U C T S I Z E C AT.# Caspase-Glo® 3/7 Assay 10 × 10ml G8093 Each system contains suﬃ cient reagents for 1,000 assays at 100µl per assay in a 96-well plate or 4,000 assays of 25µl per assay in a 384-well plate. Includes:• 10 × 10ml Caspase-Glo® 3/7 Buﬀ er• 10 bottles Caspase-Glo® 3/7 Substrate (lyophilized)P R O D U C T S I Z E C AT.# Caspase-Glo® 3/7 Assay 100ml G8092 Each system contains suﬃ cient reagents for 1,000 assays at 100µl per assay in a 96-well plate or 4,000 assays of 25µl per assay in a 384-well plate. Includes:• 1 × 100ml Caspase-Glo® 3/7 Buﬀ er• 1 bottle Caspase-Glo® 3/7 Substrate (lyophilized)Storage Conditions: Store the Caspase-Glo® 3/7 Substrate and Caspase-Glo® 3/7 Buﬀ er at –20°C protected from light. The Caspase-Glo® 3/7 Buﬀ er may be thawed and stored at 4°C for 3 months or at room temperature for up to4 days with no loss in signal. See the product label for expiration date.4Promega CorporaƟ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 TB323 · Revised 12/12 Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 5 TB323 · Revised 12/124059M A 03_3ACaspase-Glo ® SubstrateCaspase-Glo ® ReagentMix~30 minutes to 3 hours.Measureluminescence.Caspase-Glo ®BufferLuminometerFigure 3. Schematic diagram of the Caspase-Glo ® 3/7 Assay protocol.6Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516TB323 · Revised 12/12 3. Reagent Preparation and Storage1. Equilibrate the Caspase-Glo ® 3/7 Buﬀ er and lyophilized Caspase-Glo ® 3/7 Substrate to room temperature before use.2.Transfer the contents of the Caspase-Glo ® 3/7 Buﬀ er bottle into the amber bottle containing Caspase-Glo ® 3/7 Substrate. Mix by swirling or inverting the contents until the substrate is thoroughly dissolved to form the Caspase-Glo ® 3/7 Reagent. Buﬀ er volumes are 2.5ml for G8090, 10ml for G8091 and G8093, and 100ml for G8092.Storage: The reconstituted Caspase-Glo ® 3/7 Reagent may be stored at 4°C for up to 3 days with no loss of activity compared to that of freshly prepared reagent. Reconstituted reagent stored at 4°C for 1 week will give a signal approximately 90% of that obtained with freshly prepared reagent, while reconstituted reagent stored at 4°C for 4 weeks will give a signal approximately 75% of that obtained with freshly prepared reagent. Reconstituted reagent that has been refrozen and stored at –20°C for 1 week will give a signal approximately 75% of that of freshly prepared reagent, and refrozen reagent stored at –20°C for 4 weeks will give a signal approximately 60% of that of freshly prepared reagent.4.Detection of Caspase-3 and -7 Activities in Cell-Based AssaysDirections are given for performing the assay in a total volume of 200µl using 96-well plates. However, the assay can be adapted to other volumes, provided the 1:1 ratio of Caspase-Glo ® 3/7 Reagent volume to sample volume is used (e.g., 25µl of sample and 25µl Caspase-Glo ® 3/7 Reagent in a 384-well format).Materials to Be Supplied by the User • white-walled multiwell plates adequate for cell culture and compatible with the luminometer being used, such asLabsystems Cliniplate • multichannel pipet or automated pipetting station • plate shaker for mixing multiwell plates • luminometer capable of reading multiwell plates 4.A. Assay ConditionsPrepare the following reactions to detect caspase-3 and -7 activities in cell culture. Grow cells in multiwell plates that are adequate for cell culture and compatible with the luminometer being used. “Vehicle” refers to the solvent used to dissolve the drug or protein of interest.• Blank reaction: Caspase-Glo ® 3/7 Reagent, vehicle and cell culture medium without cells •Negative control: Caspase-Glo ® 3/7 Reagent and vehicle-treated cells in medium• Assays: Caspase-Glo ® 3/7 Reagent and treated cells in mediumThe blank reaction is used to measure background luminescence associated with the cell culture system and Caspase-Glo ® 3/7 Reagent. Subtract the value for the blank reaction from experimental values. Negative control reactions are important for determining the basal caspase activity of the cell culture system. Because of the sensitivityPromega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 7 TB323 · Revised 12/12of this assay, background caspase activity can be detected in serum as well as in untreated cells. Perform positive and negative controls for each plate when processing multiple assay plates.Due to the sensitive nature of the Caspase-Glo ® 3/7 Assay, take care to avoid contamination with solutions containing caspase enzymes or luciferin.Notes:1. You may need to determine empirically the optimal cell number, apoptosis induction treatment and incubation time for the cell culture system. We recommend using <20,000 cells/well in a 96-well plate.2. Use identical cell number and volume for the assay and the negative control samples.3.Total incubation time for the assay depends upon the culture system, but typically peak luminescent signal will be reached in 1–2 hours. For optimal results, the maximum recommended incubation time is 3 hours. In general the luminescent signal remaining at 3 hours is greater than 70% of peak luminescence.10188MAS i g n a l -t o -N o i s e R a t i oNumber of CellsCaspase-Glo ®Assay, Treated Caspase-Glo ®Assay, UntreatedRhodamine-110 Substrate, Treated Rhodamine-110 Substrate, UntreatedFigure 4. A comparison between bioluminescent and ﬂ uorescent caspase assays. HeLa cells were grownin OptiMEM ®(Gibco) +1% fetal bovine serum (Hyclone). Cells were plated in 96-well plates and treated withstaurosporine (1μM) for 4.5 hours or left untreated. Caspase-Glo ® 3/7 Reagent and Rhodamine-110 Substrate were added to wells, and luminescence and ﬂ uorescence were recorded at one hour on a GloMax ® Multi+ System using the Blue (490nm Ex , 510–570nm Em ) optical kit. Results were plotted as signal-to-noise ratios. Background readings weredetermined from wells containing culture medium without cells.8Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516TB323 · Revised 12/12 4.A. Assay Conditions (continued)10185M AL u m i n e s c e n c e (R L U ), B a c k g r o u n d S u b s t r a c t e dCell Number101001,00010,000100,0001,000,00010,000,0001101001,00010,0000.5-hour read 1-hour read 2-hour read 3-hour readA.B.L u m i n e s c e n c e (R L U ), B a c k g r o u n d S u b s t r a c t e dCell NumberFigure 5. Sensitivity and signal stability of caspase activity in apoptotic Jurkat cells. Jurkat cells weretreated with anti-Fas mAb (14,15) for 4.5 hours to induce apoptosis; an identical population of cells was left untreated. The Caspase-Glo ® 3/7 Reagent was added directly to cells in 96-well plates; the ﬁ nal volume was 200μl per well. The assays were incubated at room temperature for various times before recording luminescence with a GloMax ® Multi+ Luminometer. Each point represents the average of 4 wells. The “no-cell” blank control value has been subtracted from each point. Panel A. Luminometer readings were taken 1 hour after adding Caspase-Glo ® 3/7 Reagent. Panel B. Luminometer readings were taken at various times after adding the Caspase-Glo ® 3/7 Reagent. Data generated with untreated cells are not shown. Note: Due to the extended dynamic range of the Caspase-Glo ® 3/7 Assay, the data for Panel B were graphed on a log scale.Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 9 TB323 · Revised 12/124.B. Standard Protocol for Cells Cultured in a 96-Well Plate 1. Before starting the assay, prepare the Caspase-Glo ® 3/7 Reagent (see Section 3). Allow the reagent to equilibrate to room temperature. Mix well.2. Remove 96-well plates containing cells from the incubator and allow plates to equilibrate to room temperature.3.Add 100µl of Caspase-Glo ® 3/7 Reagent to each well of a white-walled 96-well plate containing 100µl of blank, negative control cells or treated cells in culture medium. Because of the sensitivity of this assay, be careful not to touch pipet tips to the wells containing samples to avoid cross-contamination. Cover the plate with a plate sealer or lid.If you are reusing pipet tips, do not touch pipet tips to the wells containing samples to avoid cross-contamination.Gently mix contents of wells using a plate shaker at 300–500rpm for 30 seconds. Incubate at room temperature for 30 minutes to 3 hours, depending upon the cell culture system. The optimal incubation period should be determined empirically.ﬂ uctuations will aﬀ ect the luminescence reading. If the room temperature ﬂ uctuates, use aconstant-temperature incubator. See Section 6.5.Measure the luminescence of each sample in a plate-reading luminometer as directed by the luminometer manufacturer.5.Detection of Caspase-3 or -7 Activity Using Puriﬁ ed CaspasesDirections are given for performing the assay in a total volume of 200µl using 96-well plates. However, the assay can be easily adapted to diﬀ erent volumes provided the 1:1 ratio of Caspase-Glo ® 3/7 Reagent volume to sample volume is used (e.g., 25µl of sample and 25µl Caspase-Glo ® 3/7 Reagent in a 384-well format).Materials to Be Supplied by the User • white-walled multiwell luminometer plates • multichannel pipet or automated pipetting station • plate shaker, for mixing multiwell plates • luminometer capable of reading multiwell plates • puriﬁ ed caspase-3 or -7 enzyme (e.g., BIOMOL Cat.# SE-169)5.A. Assay ConditionsPrepare the following reactions in luminometer plates to detect caspase-3 or -7 activity or the inhibition of activity in puriﬁ ed enzyme preparations. “Vehicle” refers to the solvent used to dissolve the test compound.• Blank reaction: Caspase-Glo ® 3/7 Reagent and vehicle control for enzyme treatment agent or inhibitor, if used • Positive control: Caspase-Glo ® 3/7 Reagent, vehicle control and puriﬁ ed caspase-3 or -7 enzyme •Experimental reactions: Caspase-Glo ® 3/7 Reagent, test compound and puriﬁ ed caspase-3 or -7 enzyme10Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-25165.A. Assay Conditions (continued)The blank reaction is used to measure background luminescence associated with the vehicle used to deliver the test compound in the presence of the Caspase-Glo ® 3/7 Reagent. Luminescence values for the blank reaction should be subtracted from experimental values. The positive control is used to determine the maximum luminescence that can be obtained with the puriﬁ ed enzyme. Positive and negative controls should be performed for each plate when processing multiple assay plates.Notes:1. You may need to determine the optimal caspase concentration and treatment vehicle empirically. In general we recommend <1ng of puriﬁ ed caspase-3 enzyme per well.2.Use identical enzyme concentrations for the assay and positive control reactions.3. Caspase spe c iﬁ c activities and unit deﬁ nitions can vary widely, depending on the manufacturer. You many need to optimize procedures describing the use of caspase by weight, depending on the speciﬁ c activity of the caspase being used.5.B. Standard Protocol for Puriﬁ ed Caspase Enzyme 1. Before starting the assay, prepare the Caspase-Glo ® 3/7 Reagent (see Section 3). Allow the reagent to equilibrateto room temperature. Mix well.2.Prepare the blank reactions, positive controls and test samples described in Section 5.A by adding 100µl of vehicle, puriﬁ ed enzyme with vehicle or puriﬁ ed enzyme with test compound to each well of a white-walled 96-well luminometer plate.If you are reusing pipet tips, do not touch the pipet tips to the wells containing samples to avoid contamination.Add 100µl of Caspase-Glo ® 3/7 Reagent to each sample. Plates may be covered with a plate sealer if incubating reactions for extended periods of time (>1 hour).ﬂ uctuations will aﬀ ect the luminescence readings. If the room temperature ﬂ uctuates, use aconstant-temperature incubator. See Section 6.4.Gently mix contents of wells using a plate shaker. Incubate at room temperature.Notes:1.The maximal luminescent signal will be reached in approximately 20–60 minutes, and this signal will be stable for several hours (see Figure 6). Peak luminescence is usually achieved sooner in assays using puriﬁ ed enzymes rather than cells. In general the luminescent signal remaining at 2 hours after peak luminescence is approximately 70% of peak luminescence. For optimal results, the maximum recommended incubation time is 3 hours.2. Gentle mixing may be done using a plate shaker. Mixing is not required but may improve reproducibility between replicate samples.5.Measure the luminescence of each sample in a plate-reading luminometer as directed by the luminometer manufacturer.4096M A 04_3A10 units/well 1 unit/well 0.1unit/well 0.01 unit/well no caspaseTime (hour)L u m i n e s c e n c e (R L U )Time (hour)L u m i n e s c e n c e (R L U)A.B.4,0008,00012,00016,00020,00010 units/well no caspaseFigure 6. Signal stability using puriﬁ ed caspase-3 enzyme. Puriﬁ ed recombinant caspase-3 enzyme was titrated and assayed in a total volume of 200µl per well of a 96-well plate using the Caspase-Glo ® 3/7 Assay. Caspase-3 was diluted in 10mM HEPES buﬀ er containing 0.1% Prionex ® as a carrier. One unit of caspase (0.1ng of protein) is the amount of enzyme required to cleave 1pmol of substrate (Ac-DEVD-pNA) hydrolyzed/minute at 30°C per the manufacturer’s unit deﬁ nition. Panel A. Data for each concentration of caspase-3 are graphed on a log graph. Panel B. Data generated using 10 units/well of caspase-3 are shown on a linear scale.12Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-25165.B. Standard Protocol for Puriﬁ ed Caspase Enzyme (continued)4060M A 03_3ASample #L u m i n e s c e n c e (R L U )+ Caspase (10u/well)– CaspaseFigure 7. Z´-factor analysis. Z´-factor values (11) for the Caspase-Glo ® 3/7 Assay were calculated using recombinant caspase-3 (10u/well) and a no-caspase blank. Assays were performed in a total volume of 200µl in two 96-well plates (80 wells/plate). Half of the wells of each plate contained buﬀ er and puriﬁ ed caspase (+), and half of the wells contained buﬀ er only with no puriﬁ ed caspase (–). Z´ factor = 0.92 for this assay [1 unit of caspase (0.1ng of protein) is the amount of enzyme required to cleave 1pmol of substrate (Ac-DEVD-pNA) hydrolyzed/minute at 30°C, per the manufacturer’s unit deﬁ nition].4988MA432C e l l V i a b i l i t y F l u o r e s c e n c e (R F U x 104)C a s p a s e A c t i v i t yL u m i n e s c e n c e (R L U )Staurosporine (µM)CellTiter-Blue ® Assay Signal Caspase-Glo ® Assay SignalB.Figure 8. Coupled increase in caspase activity and decrease in cell viability measured within the same well. HEK 293 cells were plated in 1536-well plates and allowed to attach for 10 hours at 37°C/5% CO 2. Apoptosis was induced by treating with various doses of staurosporine for 16 hours at 37°C/5% CO 2. CellTiter-Blue ® Reagent was diluted 1:4 in PBS and added to the plates by the Equator™ HTS workstation during the ﬁ nal two hours of incubation. Fluorescence units were recorded, and Caspase-Glo ® 3/7 Reagent was added by the Equator™ HTS workstation. Plates were incubated at room temperature, and luminescence was recorded using a BMG LABTECH PHERAstar Microplate Reader.14Promega Corpora Ɵ on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-25166. General Considerations SensitivityIn ﬂ uorescence-based assays, ﬂ uorescent substrates, such as Z-DEVD-AMC, are cleaved by the protease, and the released ﬂ uor exhibits a shift in the excitation/emission wavelengths. Consequently, there can be some overlap in the emission spectra of the substrate before and after cleavage, creating substantial inherent background. The luminogenic substrate (Z-DEVD-aminoluciferin) is not a substrate for luciferase until cleaved to release aminoluciferin; hence there is insigniﬁ cant inherent background (Figure 4).The low background results in a high signal:background ratio (see Figure 7). The low background also allows a broad range of linearity for the assay, approximately 4 orders of magnitude of caspase concentration (see Figure 2), andallows detection of caspase activity in as few as 20 apoptotic cells (see Figure 4). Because of the high signal:background ratio and the broad range of linearity, typically, the data need to be plotted on a log-scale graph (see Figures 2–7). We recommend using cell numbers <20,000 cells/well in a 96-well plate or puriﬁ ed caspase-3 enzyme at <1ng/well to maintain maximum signal stability.Because of the sensitivity of this assay, caspase-3 and -7 activities can be detected in fetal bovine, horse and calf serum. The luminescent signals generated from serum in the assay were inhibited by the caspase inhibitors Ac-DEVD-CHO and Z-VAD-FMK in a dose-dependent manner. When performing an assay using cells in medium with serum, we strongly recommend a “no-cell” control in addition to an untreated control to account for the signal generated from serum alone. The “no-cell” control signal can be subtracted from signal produced by the treated and untreated cells.Unlike ﬂ uorescence-based assays, the maximum sensitivity of the Caspase-Glo ® 3/7 Assay is not dependent uponextended incubations to accumulate cleaved product. Therefore, maximum sensitivity is achieved once the caspase and luciferase activities reach steady state. Typically this occurs within one hour. The time required for the assay to reach steady state may vary, depending upon the cell culture system used.TemperatureThe intensity and rate of decay of the luminescent signal from the Caspase-Glo ® 3/7 Assay depends on the rate of decrease in caspase activity and the rate of the luciferase reaction. Environmental factors that aﬀ ect the rate of the luciferase reaction also will aﬀ ect the intensity of light output and the stability of the luminescent signal. Temperature can aﬀ ect the rate of this enzymatic assay and thus the light output. For consistent results, equilibrate assay plates to a constant temperature before performing the assay. For batch-mode processing of multiple assay plates, positive and negative controls should be included for each plate. Additionally, precautions should be taken to ensure complete temperature equilibration. Plates removed from a 37°C incubator and placed in tall stacks at room temperature will require longer for equilibration than plates arranged in a single layer.。