Refresher_Stem_Cells
分子生物学
单亚基的因子（35 kD） 能把TFIID与TFIIF/Pol II相连在一起，即是聚合
酶II结合到预起始复合物所必需的 能与一些基因特异转录因子相互作用，促进转录
四、真核生物的基因转录及其调控
4. 真核生物的通用转录因子 （1）II类因子（class II factors） TFIIF的结构及功能
TFIIA
在酵母中有2个亚基，在果蝇和人中有3个亚基 TFIIA可以看成是一种TAFII（与TBP结合，
能稳定TFIID与启动子之间的结合） 在体外体系中，TFIIA并非必不可少
四、真核生物的基因转录及其调控
4. 真核生物的通用转录因子 （1）II类因子（class II factors） TFIIA与TFIIB的结构及功能
四、真核生物的基因转录及其调控
4. 真核生物的通用转录因子 （1）II类因子（class II factors） TFIIE和TFIIH的结构及功能
TFIIH
最后一个结合到预起始复合物的通用转录因子， 结构、功能均复杂
功能之一是使Pol II最大一个亚基的羧基末端域 （CTD）磷酸化，即使Pol IIA变为Pol IIO，从 而导致转录起始到转录延伸过渡
有些基因甚至没有TATA区
看家基因（housekeeping genes） 控制发育的基因
四、真核生物的基因转录及其调控
2. 真核RNA聚合酶识别的启动子 （1）RNA聚合酶II识别的启动子 起始子（initiator）
转录起始位点前后的保守序列 共同序列为：PyPyANT/APyPy
分子生物学
四、真核生物的基因转录及其调控
2. 真核RNA聚合酶识别的启动子 （1）RNA聚合酶II识别的启动子（II类启动子，
系统消息sib-mib
系统消息sib-mib系统信息⼴播详解（⼀）⼀、TD-SCDMA中各系统信息块简介（以下说明都是以3GPP R5版本为参考。
由于涉及消息IE较多，本⽂不再列出，阅读本⽂可参照3GPP 25.331相应消息结构，下同。
）1、主信息块MIB：包括MIB Value Tag，⽀持的PLMN类型，PLMN ID，以及关于其他SIBs和SB的调度信息。
2、SB1和SB2：其出现决定于MIB中的调度信息，SB的作⽤也是承载其他SIBs的调度信息。
3、SIB1：包括NAS系统信息，UE在空闲态和连接态下所使⽤的定时器和常数信息。
4、SIB2：URA ID信息。
5、SIB3：⼩区选择和重选的参数，包括Cell identity、Cell selection and re-selection info 和Cell Access Restriction三个信息IE。
下⾯对这些IE的内容进⾏深⼊剖析。
在IE Cell selection and re-selection info中，包含了以下⼀些⽤于⼩区选择和重选的参数：(1) Sintrasearch和Sintersearch⽤于进⾏同频/异频⼩区重选时，判断是否进⾏同频/异频⼩区重选的门限参数。
当TD主⼩区的S值⼩于等于Sintrasearch时，就要执⾏同频⼩区重选测量；另外如果此Sintrasearch参数没有在系统消息内部⼴播，也要执⾏同频⼩区重选测量。
同理，当TD主⼩区的S值⼩于等于Sintersearch时，就要执⾏异频⼩区重选测量；另外如果此Sintersearch参数没有在系统消息内部⼴播，也要执⾏异频⼩区重选测量。
(2) 参数Qrxlevmin、Qhyst1s和Qhyst2s⽤于进⾏⼩区选择S准则和⼩区重选排序R准则的公式计算，其中Qhyst1s和Qhyst2s⽤于UE处于IDLE状态，Qhyst1s,PCH和Qhyst2s,PCH⽤于UE处于CELL_PCH 状态，Qhyst1s,FACH和Qhyst2s,FACH⽤于UE 处于CELL_FACH状态。
仪器SOP文件——检验科各种仪器操作规程
仪器SOP文件--检验科各种仪器操作规程仪器 SOP 文件文件编号:ABCD—2-0130 第A版编制：审核:批准：生效日期:2011 年 1 月 1 日涿州市医院检验科文件编号: 涿州市医院检验科版本/修订号：A/0 生效日期:20110101 主题内容仪器 SOP 文件第 1 页共 253 页目录UD—S 流式全自动染色尿沉渣仪操作规程.。
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.7BIO1500—?—B2 型生物安全柜的标准操作程序...。
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8CyberScan pH 510 型台式 pH /Mv 仪标准操作程序.。
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.. 11AE500 电子天平操作程序.。
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..14TDL-60B 型低速台式离心机的标准操作程序。
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jade教程
基本功能
Edit菜单： Preference命令：设置显示、仪器、报告和个性化参数 尤其是仪器半高宽曲线的设置，在计算晶粒尺寸和微观应变时都将用到这个参数；
基本功能
Report菜单： 显示/打印/保存各种处理后的报告。例如：寻峰，物相检索，精修，拟合，经理尺寸计算，微观 应变计算等。可以直接打印也可以保存为文件。
常用工具栏作用
打开文件
寻峰
拟合 物相检索
打印预览
平滑图谱
手动寻峰 编辑背景线
手动拟合
扣除背景
查找PDF 卡片
计算峰 面积
删除峰
扣除背景
扣除背景：由于样品荧光等原因，在做图谱处理前需要先做背景扣除，左击“BG”出现一条背景 线，可以用手动工具中的“BE”调节背景线位置，调整好后，再次点击“BG”即可将背景线以下面积 扣除。右击“BG”弹出对话框可以选择背景线线型-通常选择Cubic Spline。
Iα=K1Cα/μ =K1 Xα / ρα （ Xα μα/ ρα + Xβ μβ / ρβ ） = K1 Xα / ρα [Xα（ μα/ ρα - μβ / ρβ ）+ μβ / ρβ ]=K分析的具体方法有：
外标法（单线条法） 内标法 K值法 直接比较法（绝热）
Save命令（将原始文件保存为txt；）
需要注意的是Save命令保存的是窗口中显示的图谱，如果窗口中显示的只有部分图谱，那么将 只会保存显示的部分，其他的将不保存。保存前注意设置范围（FullRange（View-Zoom Window菜单）） 如果进行过平滑处理（Smooth），将保存smooth数据非原始数据；
Agilent 1290 6400 LCMS Training(液质培训)
Dionex UltiMate 3000 系列WPS自动进样器
UltiMate 3000 系列自动进样器操作手册戴安中国有限公司技术服务中心2006.5目 录1. 概述 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 22. 仪器构造 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 63. 安装 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 134. 用户界面 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 275. 软件控制 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 366. 故障分析及解决 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 487. 日常维护 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 598. 技术信息 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 819. 附件及配件 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 8210. 附录 ﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 871． 概述1.1 开箱自动进样器所有的电子元器件在出厂运输前都经过严格测试，开箱之后，请检查仪器是否在运输过程中有机械损伤。
若有请立即联系Dionex及保留原始包装材料，以便返厂运输。
打开自动进样器，按下面步骤：1．把运输包装箱放置在地板上，拿出配件工具包和电源线。
2．抓住自动进样器侧面，缓慢从包装箱中拿出仪器，放置在稳定的台面上。
自动进样器重量超过20kg（44Ibs），为了防止搬运仪器时 坠落，单个人最好不要搬运自动进样器。
3．除去泡沫和聚乙烯包装。
4．检查装箱单和订货单是否与货物相符。
为了保护进样针臂在运输过程中的安全插入了一块泡沫，在第一次使用时，除去泡沫。
1．2 仪器特点自动进样器是一种高质量的仪器，它专为 Dionex UltiMate 3000 系统的HPLC分析而设计。
它能提供高效性和重复性，甚至应用于很低进样体积的HPLC。
设计时已经优化了最小死体积和最大效率，它能用于日常分析和复杂研究任务。
z圆盘传送带能操纵三种样品固定器，每一种能带有1.8ml 40个小瓶或4ml 22个小瓶，也可选择三种标准96－孔板或384－孔板，深孔板也能很好使用。
在同一个圆盘传送带上能一起使用孔板、深孔板和小瓶。
此外，圆盘传送带还能固定额外的10ml 3×5个小瓶，例如试剂小瓶。
5G无线网络高精度仿真指导意见
另外，需要注意的是 5G 在 3.5GHz 频段是 TDD 制式，需要配置上下行时隙， 根据集团的要求和外场试验网的配置要求，仿真中设置为 2.5ms 双周期，即 DDDSU DDSUU。
基于仿真性能以及操作界面友好等因素考虑，Forsk 公司的 Atoll 应用较广泛， 且通过实际应用验证，其仿真效果也较具参考性。在 2018 年底，Forsk 公司推出 了 5G 仿真模块，本指导意见以 Atoll 软件介绍 5G 仿真方法和流程。
2.2 仿真操作步骤
一般的仿真操作步骤如下流程图所示。
2
2 仿真软件简介
5G 无线网仿真指导意见
2.1 主流仿真软件
在 LTE 时代，各大厂家都有不同的仿真软件平台用于实现 4G 制式的网络指 标预测，例如 Forsk 公司的 Atoll，Aircom 公司的 ASSET，还有华为的 UNET、爱 立信采用的 Planet、中国移动集团设计院自行研发的 ANPOP 等。
2.3.1 SPM 模型
SPM（Standard Propagation Model，标准传播模型）是从 Planet 中承袭过来 的 K 参数模型，起源为 Hata 模型，是经过大量测试数据调整得出的经验统计模
型。其传播公式如下：
L = K + K lg(d ) + K H + K lg(H ) + K lg(H ) + K lg(H )lg(d )
i
1 仿真概述
5G 无线网仿真指导意见
1.1 仿真原理及作用
仿真是通过仿真软件，使用数字地图、基站工程参数、测试数据建立网络模 型，通过系统的模拟运算得出网络覆盖预测、干扰预测及容量评估结果。主要应 用于网络规划、建设、优化阶段网络性能预测、趋势预测，为网络规划、建设、 优化提供参考。
Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Fa
实验摘要
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contentsinto oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc,and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
Invitrogen Neon
Neon™ Transfection SystemFor transfecting mammalian cells, including primary and stem cells, with high transfection efficiency. Catalog Numbers MPK5000, MPK1025, MPK1096, MPK10025, MPK10096Doc. Part No. 25-1056 Pub. No. MAN0001632 Rev.B.0WARNING! For safety and biohazard guidelines, see the “Safety” appendix in the Neon™ Transfection System User Guide (Pub.No. MAN0001557). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.Note: This Quick Reference is intended as a benchtop reference for experienced users of the Neon™ Transfection System User Guide (Pub. No. MAN0001557). For detailed instructions, supplemental procedures, and troubleshooting, see the Neon™ Transfection System User Guide (Pub. No. MAN0001557).General guidelines•Prepare high-quality plasmid DNA at a concentration of 1 to 5 μg/μL in deionized water or TE buffer, or high quality RNAi duplex at a concentration of 100–250 μM in nuclease-free water.•Use an appropriate GFP (green fluorescent protein) construct or siRNA control to determine transfection efficiency. See the Neon™Transfection System User Guide (Pub. No. MAN0001557) for details.•Use Resuspension Buffer R for established adherent and suspension cells, as well as primary adherent cells. Use Resuspension Buffer T with high voltage protocols of 1900 V or more. If arcing occurs with Resuspension Buffer R, consider switching toResuspension Buffer T.•Based on your initial results, you may need to optimize the electroporation parameters for your experiment using an 18-well or pre-programmed 24-well optimization protocol.•Discard the Neon™ Tips after 2 usages and Neon™ Tubes after 10 usages as a biological hazard. Change the tube and buffer when switching to a different plasmid DNA/siRNA or cell type.•The volume of plasmid DNA or siRNA added to the tranfection reaction should not exceed 10% of the total transfection volume.•Visit for a library of electroporation protocols for a variety of commonly used cell types.Prepare cellsFor the appropriate volume of medium to use based on cell density, or plating volumes for other plate formats, see “Amount of reagents” on page 2.1.Cultivate the required number of cells (70% to 90%confluent on the day of transfection) by seeding a flaskcontaining fresh growth medium 1 to 2 days prior toeletroporation.2.On the day of the experiment, pre-warm aliquots of culturemedium containing serum, PBS (without Ca2+and Mg2+), and Trypsin/EDTA solution to 37°C.3.Rinse the cells with PBS (without Ca2+and Mg2+), thentrypsinize the cells with the Trypsin/EDTA solution.4.Take an aliquot of trypsinized cell suspension, then countcells to determine the cell density.5.Harvest the cells in growth medium containing serum.6.Transfer cells to a 1.5-mL microcentrifuge tube or a 15-mLconical tube, then centrifuge the cells at 100 - 400 × g for 5 minutes at room temperature.7.Wash cells with PBS (without Ca2+and Mg2+) bycentrifugation at 100 - 400 × g for 5 minutes at roomtemperature.8.Aspirate the PBS, then resupsend the cell pellet inResuspension Buffer R (or Resuspension Buffer T forprograms ≥ 1900 V) at a final density of 1.0 × 107 cells/mL for adherent cells or 2.0 × 107 cells/ml for suspension cells.Gently pipette the cells to obtain a single cell suspension.IMPORTANT! Avoid storing the cell suspension for more than 15 to 30 minutes at room temperature. This will reduce cell viability and transfection efficiency.9.Prepare 24-well plates by filling the wells with 500 μL ofculture medium containing serum and supplements, butwithout antibiotics. Pre-incubate plates in a 37°C, 5% CO2 humidified incubator.Amount of reagentsFor each electroporation sample, the amount of plasmid DNA/siRNA, cell number, and volume of plating medium per well are listed in the following table. Use Resuspension Buffer T for cell types that require high voltage protocols of 1900 V or more. For all other cell types, use Resuspension Buffer R.[1]Use Resuspension Buffer T for primary suspension blood cells.Using the Neon ™Transfection SystemFor details on setting up the Neon ™device and Neon ™PipetteStation, see the Neon ™Transfection System User Guide (Pub. No.MAN0001557).1.Select the appropriate protocol for your cell type. Use one ofthe following options:•Input the electroporation parameters in the Input window if you already have the electroporation parameters for your cell type.•Tap Database , then select the cell-specificelectroporation parameters that you have added for various cell types.•Tap Optimization to perform the optimization protocol for your cell type.2.Fill the Neon ™Tube with 3 mL of Electrolytic Buffer (useBuffer E for the 10 μL Neon ™Tip and Buffer E2 for the 100μL Neon ™Tip).Note: Make sure that the electrode on the side of the tube is completely immersed in buffer. 3.Insert the Neon ™ Tube into the Neon ™Pipette Station untilyou hear a click sound (Figure 1).Figure 1 Schematic of Neon ™ Tube and Neon ™ Pipette Station.4.Transfer the appropriate amount of plasmid DNA/siRNA intoa sterile, 1.5 mL microcentrifuge tube.5.Add cells to the tube containing plasmid DNA/siRNA, thengently mix. See “Amount of reagents” on page 2 for cell number, DNA/siRNA amount, and plating volumes to use.6.To insert a Neon ™Tip into the Neon ™Pipette, press thepush-button on the pipette to the second stop to open the clamp.7.Insert the top-head of the Neon ™Pipette into the Neon ™Tipuntil the clamp fully picks up the mount stem of the piston (Figure 2).Figure 2 Schematic of Neon ™ Pipette and Neon ™Tip.8.Gently release the push-button, continuing to apply adownward pressure on the pipette, ensuring that the tip is sealed onto the pipette without any gaps.9.Press the push-button on the Neon ™Pipette to the first stopand immerse the Neon ™Tip into the cell-DNA/siRNA mixture.Slowly release the push-button on the pipette to aspirate thecell-DNA/siRNA mixture into the Neon ™Tip (Figure 3).Figure 3 Schematic of Neon ™Tip.Note: Avoid air bubbles during pipetting as air bubbles cause arcing during electroporation leading to lowered or failed transfection. If you notice air bubbles in the tip,discard the sample, then carefully aspirate the fresh sample into the tip again without any air bubbles.10.Insert the Neon ™Pipette with the sample vertically into theNeon ™ Tube placed in the Neon ™Pipette Station until youhear a click sound (Figure 4).Figure 4 Schematic of Neon ™ Tube and Neon ™ Pipette Station.Note: Ensure that the metal head of the Neon ™pipette projection is inserted into the groove of the pipette station.11.Ensure that you have selected the appropriateelectroporation protocol, then press Start on the touchscreen.12.The Neon ™device automatically checks for the properinsertion of the Neon ™ Tube and Neon ™Pipette before delivering the electric pulse.13.After delivering the electric pulse, Complete is displayed onthe touchscreen to indicate that electroporation is complete.14.Slowly remove the Neon ™Pipette from the Neon ™PipetteStation. Immediately transfer the samples from the Neon ™Tip by pressing the push-button on the pipette to the first stop into the prepared culture plate containing prewarmed medium with serum and supplements but without antibiotics.Note: Discard the Neon ™ Tip into an appropriate biologicalhazardous waste container. To discard the Neon ™Tip, press the push-button to the second stop into an appropriate biological hazardous waste container.15.Repeat step 6 to step 14 for the remaining samples.Note: Be sure to change the Neon ™Tips after using it twiceand Neon ™ Tubes after 10 usages. Use a new Neon ™Tip andNeon ™Tube for each new plasmid DNA sample.16.Gently rock the plate to ensure even distribution of the cells.Incubate the plate at 37℃ in a humidified CO 2 incubator.17.If you are not using the Neon ™device, turn the power switchon the rear to OFF .18.Assay samples to determine the transfection efficiency(e.g., fluorescence microscopy or functional assay) or geneknockdown (for siRNA).19.Based on your initial results, you may need to optimizedthe electroporation parameters for your cell type. For more information, see the Neon™ Transfection System User Guide (Pub. No. MAN0001557).Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USAFor descriptions of symbols on product labels or product documents, go to /symbols-definition.The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified./support | /askaquestion。
四川省达州市外国语学校2022-2023学年高二下学期5月月考英语试题
四川省达州市外国语学校2022-2023学年高二下学期5月月考英语试题一、短对话1．Why does the woman invite the man?A．To practice English.B．To meet her parents.C．To do homework together.2．What is the man interested in?A．The postcard.B．The letter.C．The advertisement.3．Where are the speakers?A．In a bookstore.B．In a library.C．In a classroom.4．Why is the woman making the call?A．To ask for sick leave for Judy.B．To check on her daughter.C．To seek medical advice.5．What are the speakers talking about?A．A travel experience.B．A weekend plan.C．A favorite destination.二、长对话听下面一段较长对话，回答以下小题。
6．What is Alice going to do?A．Give Bill a surprise.B．Take Bill to a party.C．Pick Bill up from work.7．Who will bring dessert?A．Susan.B．Joe.C．Jim.听下面一段较长对话，回答以下小题。
8．What are the speakers discussing?A．A car accident.B．A car theft.C．A car park.9．What is said about Howard’s car?A．It was recently purchased.B．It is not insured.C．It is a good deal.听下面一段较长对话，回答以下小题。
SIMATIC 过程控制系统 PCS 7 维护站 V9.1 功能手册说明书
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T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects
DOI: 10.1126/scitranslmed.3002842, 95ra73 (2011);3 Sci Transl Med , et al.Michael Kalos and Can Establish Memory in Patients with Advanced Leukemia T Cells with Chimeric Antigen Receptors Have Potent Antitumor EffectsEditor's Summarythe potential for CAR-modified T cells to bring cancer therapy up to speed.treatment had complete remission of their leukemia. Although this is early in the clinical study, these results highlight scale with a second exposure to CLL cells. Indeed, two of the three CLL patients who underwent the CAR T cell CAR T cells persisted with a memory phenotype, which would allow them to respond more quickly and on a larger these CAR T cells expanded >1000-fold, persisted for more than 6 months, and eradicated CLL cells. Some of these allowing for much broader cellular targeting than is obtained with normal T cells. After transfer into three CLL patients,receptor could activate T cells in response to CD19 in the absence of major histocompatibility complex restriction, specific intracellular signaling domain. The resulting chimeric −specific costimulatory domain and a T cell −both a T cell bind in a restricted manner to the CD19 protein (which is found solely on normal B cells and plasma cells) attached to The CAR T cells used in this study expressed an antigen receptor that consists of antibody binding domains that as reflected by decreased numbers of B cells and plasma cells and the development of hypogammaglobulinemia.tumor cells after transfer into patients; they also mediated cancer remission. Innocent bystanders were also targeted, chronic lymphocytic leukemia (CLL) (a B cell cancer). The designer T cells not only expanded, persisted, and attacked modified T cells to express a chimeric antigen receptor (CAR) to yield so-called CAR T cells that specifically target . have genetically et al cells to the tumor and maintaining these cells in patients remains challenging. Now, Kalos harness the power of the immune system to fight cancers such as leukemia; however, targeting functional immune T to healthy tissues, such as infection or cancer, and then try to deter dangerous activity. Researchers have long sought As members of the body's police force, cells of the immune system vigilantly pursue bad actors that harmGo CAR-Ts in the Fast Lane/content/3/95/95ra73.full.html can be found at:and other services, including high-resolution figures,A complete electronic version of this article /content/suppl/2011/08/08/3.95.95ra73.DC1.htmlcan be found in the online version of this article at: Supplementary Material/about/permissions.dtl in whole or in part can be found at:article permission to reproduce this of this article or about obtaining reprints Information about obtaining last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except the Science Translational Medicine o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mL E U K E M I AT Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced LeukemiaMichael Kalos,1,2*Bruce L.Levine,1,2*David L.Porter,1,3Sharyn Katz,4Stephan A.Grupp,5,6 Adam Bagg,1,2Carl H.June1,2†Tumor immunotherapy with T lymphocytes,which can recognize and destroy malignant cells,has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens.Chimeric antigen receptors(CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens;however,to date,lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials.We report that CAR T cells that target CD19and contain a costimulatory domain from CD137and the T cell receptor z chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia(CLL).The engineered T cells expanded>1000-fold in vivo,trafficked to bone marrow,and continued to express functional CARs at high levels for at least6months.Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia.On average,each infused CAR-expressing T cell was calculated to eradicate at least1000CLL cells.Furthermore,a CD19-specific immune re-sponse was demonstrated in the blood and bone marrow,accompanied by complete remission,in two of three patients.Moreover,a portion of these cells persisted as memory CAR+T cells and retained anti-CD19effector functionality,indicating the potential of this major histocompatibility complex–independent approach for the ef-fective treatment of B cell malignancies.INTRODUCTIONUsing gene transfer technologies,T cells can be genetically modified to stably express antibody binding domains on their surface that con-fer novel antigen specificities that are major histocompatibility com-plex(MHC)–independent.Chimeric antigen receptors(CARs)are an application of this approach that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-z chain or Fc g RI protein into a single chimeric protein(1,2). Trials testing CARs are presently under way at a number of academic medical centers(3,4).In most cancers,tumor-specific antigens are not yet well defined,but in B cell malignancies,CD19is an attractive tumor target.Expression of CD19is restricted to normal and malig-nant B cells(5),and CD19is a widely accepted target to safely test CARs.Although CARs can trigger T cell activation in a manner sim-ilar to an endogenous T cell receptor,a major impediment to the clin-ical application of this technology to date has been the limited in vivo expansion of CAR+T cells,rapid disappearance of the cells after in-fusion,and disappointing clinical activity(4,6).CAR-mediated T cell responses may be further enhanced with ad-dition of costimulatory domains.In a preclinical model,we found that inclusion of the CD137(4-1BB)signaling domain significantly increased antitumor activity and in vivo persistence of CARs com-pared to inclusion of the CD3-z chain alone(7,8).To evaluate the safety and feasibility for adoptive transfer of T cells gene-modified to express such CARs,we initiated a pilot clinical trial using autologous T cells expressing an anti-CD19CAR including both CD3-z and the 4-1BB costimulatory domain(CART19cells)to target CD19+malig-nancies.To date,we have treated three patients under this protocol. Some of the findings from one of these patients are described in(9), which reports that this treatment results in tumor regression,CART19 cell persistence,and the unexpected occurrence of delayed tumor lysis syndrome.Here,we show that the CART19cells mediated potent clinical antitumor effects in all three patients treated.On average,each infused CAR T cell and/or their progeny eliminated more than 1000leukemia cells in vivo in patients with advanced chemotherapy-resistant chronic lymphocytic leukemia(CLL).CART19cells underwent robust in vivo T cell expansion,persisted at high levels for at least6 months in blood and bone marrow(BM),continued to express func-tional receptors on cells with a memory phenotype,and maintained anti-CD19effector function in vivo.RESULTSClinical protocolThree patients with advanced,chemotherapy-resistant CLL were enrolled in a pilot clinical trial for CART19cell therapy.Figure1presents a summary of the manufacturing process for the gene-modified T cells (A)and the clinical protocol design(B).All patients were extensively pretreated with various chemotherapy and biologic regimens(Table1). Two of the patients had p53-deficient CLL,a deletion that portends poor response to conventional therapy and rapid progression(10). Each of the patients had a large tumor burden after the preparative1Abramson Cancer Center,University of Pennsylvania,Philadelphia,PA19104,USA.2De-partment of Pathology and Laboratory Medicine,University of Pennsylvania,Philadelphia, PA19104,USA.3Department of Medicine,University of Pennsylvania,Philadelphia,PA 19104,USA.4Department of Radiology,University of Pennsylvania,Philadelphia,PA19104, USA.5Department of Pediatrics,University of Pennsylvania,Philadelphia,PA19104,USA. 6Division of Oncology,Children’s Hospital of Philadelphia,Philadelphia,PA19104,USA.*These authors contributed equally to this work.†To whom correspondence should be addressed.E-mail:cjune@ o n F e b r u a r y 2 0 , 2 0 1 2 s t m . s c i e n c e m a g . o r g D o w n l o a d e d f r o mchemotherapy,including extensive BM infiltration(40to95%)and lymphadenopathy;UPN02also had peripheral lymphocytosis.There was a low abundance of T cells in the apheresis products(2.29to4.46%) (table S1)as well as likely impaired T cell activation,as has been shown previously in CLL patients(11).Additional details of the cell manufac-turing and product characterization for the CART19cell preparation for each patient are shown in table S1.All patients were pretreated1to 4days before CART19cell infusions with lymphodepleting chemo-therapy(Table1).A split-dose cell infusion schedule was used to address potential safety concerns related to the evaluation of a previously untested CAR that incorporated the4-1BB costimulatory signaling domain. In vivo expansion,persistence,and BM trafficking of CART19cellsOur preclinical data in two animal models,including mice bearing xenografts of primary human precursor-B acute lymphoblastic leuke-mia(7,8),indicated that CAR+T cells that express a4-1BB signaling domain expanded after stimulation with anti-CD3/anti-CD28mono-clonal antibody–coated beads(12)and had improved persistence com-pared to CAR+T cells lacking4-1BB.We developed a quantitative polymerase chain reaction(qPCR)assay to enable quantitative tracking of CART19cells in blood and BM.CART19cells expanded and persisted in the blood of all patients for at least6months(Fig.2, A and B).Moreover,CART19cells expanded1000-to10,000-fold in the blood of patients UPN01and03during the first month after infusion,reaching peak frequencies of10to>95%of circulating white blood cells in UPN01and03(Fig.2C).The peak expansion levels coincided with onset of the clinical symptoms after infusion in UPN01 (day15)and UPN03(day23).Furthermore,after an initial decay,which can be modeled with first-order kinetics,the CART19cell numbers stabilized in all three patients from days90to180after infusion (Fig.2B).The CART19cells also trafficked to the BM in all patients, albeit in5-to10-fold fewer numbers than observed in blood(Fig.2D). CART19cells had a log-linear decay in the BM in UPN01and03, with a disappearance half-life of~35days.Induction of specific immune responses in the peripheral blood and BM compartments after CART19infusion Peripheral blood(PB)and BM serum samples from all patients were collected and batch-analyzed to quantitatively determine cytokine levels.A panel of30cytokines,chemokines,and other soluble factors were assessed for potential toxicities and to provide evidence of CART19cell function.The full data set for all of the cytokines measured in each of the three patients through the date of thisapheresisSeed in gas-permeable bags.Transduction w/αCD19-41BBζvectorVector washout.Culture in gas-permeable bagsCulture in WAV EbioreactorHarvest, wash, concentrateCryopreserve final product ininfusible cryomediaCD3/28-positive selection ofT cells with anti-CD3/anti-CD28 mAb-coated magneticbeadsDay 0Day 0-1Day 3Day 5Harvest day(10 ±2)ABManufacture/cryopreservationFig.1.Schematic representation of the gene transfer vector and trans-gene,gene-modified T cell manufacturing,and clinical protocol design.(A)T cell manufacturing.Autologous cells were obtained via leukapher-esis,and T cells were enriched by mononuclear cell elutriation,washed, and expanded by addition of anti-CD3/CD28–coated paramagnetic beads for positive selection and activation of T cells.Residual leukemic cells were depleted.The lentiviral vector was added at the time of cell activation and was washed out on day3after culture initiation.Cells were expanded on a rocking platform device(WAVE Bioreactor System) for8to12days.On the final day of culture,the beads were removed by passage over a magnetic field and the CART19cells were harvested and cryopreserved in infusible medium.mAb,monoclonal antibody.(B)Clin-ical protocol design.Patients were given lymphodepleting chemo-therapy as described,followed by CART19infusion#1by intravenous gravity flow drip over a period of15to20min.The infusion was given using a split-dose approach over3days(10,30,and60%)beginning1to5days after completion of chemotherapy.Endpoint assays were conducted on study week4.At the conclusion of active monitoring, subjects were transferred to a destination protocol for long-term follow-up as per FDA guidance.onFebruary2,212stm.sciencemag.orgmpublication is presented in tables S2to S5.Of the analytes tested, 11had a threefold or more change from baseline,including four cytokines[interleukin-6(IL-6),interferon-g(IFN-g),IL-8,and IL-10], five chemokines[macrophage inflammatory protein–1a(MIP-1a), MIP-1b,monocyte chemotactic peptide–1(MCP-1),CXC chemokine ligand9(CXCL9),and CXCL10],and the soluble receptors IL-1R a and IL-2R a;IFN-g had the largest relative change from baseline (Fig.3).The peak time of cytokine elevation in UPN01and03 correlated temporally with both the previously described clinical symptoms and the peak levels of CART19cells detected in the blood for each patient.Notably,cytokine modulations were transient,and levels reverted to baseline relatively rapidly despite continued func-Table1.Patient demographics and response.CR,complete response;PR,partial response;N/A,not available.Subject UPNAge/sexkaryotypePrevious therapiesCLL tumor burden at baselineTotaldoseof CART19(cells/kg)Response day+30(duration)BM(study day)‡Blood(studyday)‡Nodes/spleen(study day)‡0165/Mnormal Fludarabine×four cycles(2002)Hypercellular70%CLL N/A 6.2×1011to1.0×1012CLL cells(day−37)1.1×109(1.6×107/kg)CR(11+months)Rituximab/fludarabine×four cycles(2005)2.4×1012CLL cells(day−14)Alemtuzumab×12weeks(2006)1.7×1012CLL cells(day−1)Rituximab(two courses,2008to2009)R-CVP×two cycles(2009)Lenalidomide(2009)PCR×two cycles(5/18/2010to6/18/2010)Bendamustine×one cycle(7/31/10to8/1/10)pre-CART190277/M del(17)(p13)*Alemtuzumab×16weeks(6/2007)Hypercellular>95%CLL2.75×1011CLL cells(day−1)1.2×1012to2.0×1012CLL cells(day−24)5.8×108(1.0×107/kg)PR(7months)Alemtuzumab×18weeks(3/2009)3.2×1012CLL cells(day−47)Bendamustine/rituximab:7/1/2010(cycle1)7/28/2010(cycle2)8/26/2010(cycle3)pre-CART190364/M del(17)(p13)†R-Fludarabine×twocycles(2002)Hypercellular40%CLLN/A 3.3×1011to5.5×1011CLL cells(day−10)1.4×107(1.46×105/kg)CR(10+months)R-Fludarabine×four cycles(10/06to1/07)8.8×1011CLL cells(day−1)R-Bendamustine×one cycle(2/09)Bendamustine×three cycles(3/09to5/09)Alemtuzumab×11weeks(12/09to3/10)Pentostatin/cyclophosphamide(9/10/10)pre-CART19*UPN02karyotype[International System for Human Cytogenetic Nomenclature(ISCN)]:45,XY,del(1)(q25),+del(1)(p13),t(2;20)(p13;q11.2),t(3;5)(p13;q35),add(9)(p22),?del(13)(q14q34),-14,del(17) (p13)[cp24].†UPN03karyotype(ISCN):46,XY,del(17)(p12)[18]/44~46,idem,der(17)t(17;21)(p11.2;q11.2)[cp4]/40~45,XY,-17[cp3].‡See the Supplementary Material for methods of tumor burden determination.o n F e b r u a r y 2 0 , 2 0 1 2 s t m . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtional persistence of CART19cells.Only modest changes in cytokine levels were noted in UPN 02,possibly as a result of corticosteroid treatment.We also noted a robust induction of cytokine secretion in the supernatants from BM aspirates of UPN 03(Fig.3D and table S5).Although a pretreatment marrow sample was not available,compared to the late time point (+176),we also observed elevated levels for a number of factors in the +28marrow sample for UPN 01including IL-6,IL-8,IL-2R,and CXCL9;in contrast,compared to the pretreatment marrow sample,no elevation in cytokines was de-tected in the +31day sample for UPN 02(table S5).One of the preclinical rationales for developing CAR +T cells with 4-1BB signaling domains was a projected reduced propensity to trigger IL-2and tumor necrosis factor –a (TNF-a )secretion compared to CAR +T cells with CD28signaling domains (7);indeed,elevated amounts of soluble IL-2and TNF-a were not detected in the serum of the patients.Lower levels of these cytokines may be related to sustained clinical ac-tivity:Previous studies have shown that CAR +T cells are potentially suppressed by regulatory T cells (13),which can be elicited by either CARs that secrete substantial amounts of IL-2or by the provision of exogenous IL-2after infusion.Moreover,the TNF-a is complicit in cy-tokine storm –related effects in patients,which are absent here.Prolonged receptor expression and establishment of a population of memory CART19cells in bloodA central question in CAR-mediated cancer immunotherapy is whether optimized cell manufacturing and costimulation domains will enhance the persistence of genetically modified T cells and permit the establishment of CAR +memory T cells in patients.Previous studies have not demonstrated robust expansion,prolonged persist-ence,or functional expression of CARs on T cells after infusion (14–17).The high persistence of CART19cells that we observed at late time points for UPN 03facilitated a more detailed phenotypic analysis ofpersisting cells.Flow cytometric analysis of samples from both blood and BM 169days after infusion revealed the presence of CAR19-expressing cells in UPN 03as well as an absence of B cells (fig.S1,Aand B).These CAR +cells persisted in allthree patients beyond 4months,as shown by qPCR (Fig.2).The in vivo frequency of CAR +cells by flow cytometry closely matched the values obtained from the PCR assay for the CAR19transgene.CAR expression was also detected on the surface of 5.7and 1.7%of T cells in the blood of patient UPN 01on days 71and286after infusion (fig.S2).We next used polychromatic flow cy-tometry to perform detailed studies and further characterize the expression,phe-notype,and function of CART19cells in UPN 03using an anti-CAR idiotype anti-body (MDA-647)and the gating strategy shown in fig.S3.We observed differencesin the expression of memory and activa-tion markers in both CD4+and CD8+T cells based on CAR19expression.In the CD4+compartment,at day 56,CART19cells were characterized by auniform lack of CCR7,a predominance of CD27+/CD28+/PD-1+cells distributed within both CD57+and CD57−compart-ments,and an essential absence of CD25and CD127expression,the latter two markers defining regulatory CD4+T cells (18)(Fig.4A).In contrast,CAR −cells at this time point were heterogeneous in CCR7,CD27,and PD-1expression;expressed CD127;and also contained a substantialCD25+/CD127−population.By day 169,although CD28expression remained uni-formly positive in all CART19CD4+cells,a fraction of the CART19CD4+cells had acquired a central memory phenotype,withA CB Day (after infusion)110100100010000100000Day (after infusion)T o t a l c e l l s i n c i r c u l a t i o nDay (after infusion)110100100010000D C o p i e s /µg g D N A% W B CDay (after infusion)C o p i e s /µg gD N AWBC and CART 19: Blood CART 19: Marrow 10101010101010Fig.2.Sustained in vivo expansion and persistence in blood and marrow of CART19cells.(A to D )qPCRanalysis was performed on DNA isolated from whole blood (A to C)or bone marrow (BM)(D)samples obtained from UPN 01,UPN 02,and UPN 03to detect and quantify CAR19sequences.The frequency of CART19cells is shown as average transgene copies (A),total calculated CART19cells in circulation (B),or as a fraction of circulating white blood cells (WBCs)(C).(A)Copies CAR19/microgram DNA is calculated as de-scribed in Materials and Methods.(B)The total number of lymphocytes (total normal and CLL cells)versus total CART19+cells in circulation is plotted for all three subjects using the absolute lymphocyte count from complete blood count values and assuming a 5.0-liter volume of peripheral blood.(C)%WBC is calculated as described in Materials and Methods.(D)Bulk qPCR analysis of marrow to quantify CART19sequences.The data from patient UPN 03in (A,C,and D)has been published in (9)and is reprinted here with permission.Each data point represents the average of triplicate measurements on 100to 200ng of genomic DNA,withmaximal percent coefficient of variation (CV)less than 1.56%.Pass/fail parameters for the assay included preestablished ranges for slope and efficiency of amplification,and amplification of a reference sample.The lower limit of quantification for the assay established by the standard curve range was two copies of transgene per microgram of genomic DNA;sample values below that number are considered estimates and presented if at least two of three replicates generated a C t value with percent CV for the values 15%.CART19cells were infused at days 0,1,and 2for UPN 01and 03and at days 0,1,2,and 11for UPN 02. o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mCCR7expression,a higher percentage of CD27−cells,the appearance of a PD-1−subset,and acquisition of CD127expression.At day 169,CAR −cells remained reasonably consistent with their day 56counterparts,with the exception of a reduction in CD27expression and a decrease in the percentage of CD25+/CD127−cells.In the CD8+compartment,at day 56,CART19CD8+cells displayed primarily an effector memory phenotype (CCR7−,CD27−,CD28−),con-sistent with prolonged and robust exposure to antigen (Fig.4B).In con-trast,CAR −CD8+T cells consisted of mixtures of effector and central memory cells,with CCR7expression in a subset of cells,and substantial numbers of cells in the CD27+/CD28−and CD27+/CD28+fractions.Al-though a large percentage of both CART19and CAR −cell populations expressed CD57,a marker associated with memory T cells with high cytolytic potential (19),this molecule was uniformly coexpressed with PD-1in the CART19cells,a possible reflection of the extensive replicative history of these cells.In contrast to the CAR −cell population,the entirety of the CART19CD8+population lacked expression of both CD25and CD127,markers associated with T cell activation and the development of functional memory cells (20).By day 169,although the phenotype of the CAR −cell population remained similar to the day 56cells,the CART19population had evolved to contain a population with features of central memory cells,notably expression of CCR7and higher levels of CD27and CD28,as well as cells that were PD-1−,CD57−,and CD127+.Effector function of CART19cells after 6months in blood In addition to a lack of long-term persistence,a limitation of previous trials with CAR +T cells has been the rapid loss of functional activity of the infused T cells in vivo.The high level of CART19cell persist-ence and surface expression of the CAR19molecule in UPN 03provided the opportunity to directly test anti-CD19–specific effector functions in cells recovered from cryopreserved PB samples.Pe-ripheral blood mononuclear cells (PBMCs)from UPN 03were cultured with target cells that either did or did not express CD19(Fig.4C and fig.S3).Robust CD19-specific effector function of CART19cells was observed by the specific degranulation of CART19cells against CD19+but not CD19−target cells,as assessed by surface CD107a expression.Notably,exposure of the CART19population to CD19+targets induced a rapid internalization of surface CAR19(see fig.S3for constitutive surface expression of CAR19in the same ef-fector cells in standard flow cytometric staining).The presence of costimulatory molecules on target cells was not required for trigger-ing CART19cell degranulation because the NALM-6line,which was used as a target in these studies,does not express CD80or CD86(21).Effector function was evident at day 56after infusion and was re-tained at day 169(Fig.4C).Robust effector function of CAR +and CAR −T cells could also be demonstrated by pharmacologic stimula-tion with phorbol 12-myristate 13-acetate (PMA)and ionomycin.ADay (after infusion)S e r u m c y t o k i n e (f o l d c h a n g e f r o m b a s e l i n e )(f o l d c h a n g e f r o m b a s e l i n e )S e r u m c y t o k i n e (f o l d c h a n g e f r o m b a s e l i n e )BCDay (after infusion)D Day (after infusion)C o n c e n t r a t i o n (p g /m l )αIL-6 IFN-γCXCL10MIP-1βMCP-1CXCL9IL-2R αIL-8 IL-10MIP-1αFig.3.Serum and BM cytokines before and afterCART19cell infusion.(A to C )Longitudinal measure-ments of changes in serum cytokines,chemokines,and receptors in UPN 01(A),UPN 02(B),and UPN 03(C)on the in-dicated day after CART19cell infusion.(D )Serial assessments of the same analytes in the BM from UPN 03.Analytes with agreater than or equal to threefold change are indicated and plotted as relative change from baseline (A to C)or as absolute values (D).In (C)and (D),a subset of the cytokine data (IFN-g ,CXCL10,CXCL9,IL-2R a ,and IL-6)from UPN 03have been pub-lished in (9)and are reprinted here with permission.Absolutevalues for each analyte at each time point were derived from a recombinant protein-based standard curve over a threefold eight-point dilution series,with upper and lower limits of quantification determined by the 80to 120%observed/expected cutoff valuesfor the standard curves.Each sample was evaluated in duplicate with average values calculated and percent CV in most cases less than 10%.To accommodate consolidated data presentation in the context of the wide range for the absolute values,data are presented as fold change over the baseline value for each analyte.In cases where baseline values were not detectable,half of the lowest standard curve value was used as the baseline value.Standard curve ranges for analytes and baseline (day 0)values (listed in parentheses sequentially for UPN 01,02,and 03),all in pg/ml:IL-1R a :35.5to 29,318(689,301,and 287);IL-6:2.7to 4572(7,10.1,and 8.7);IFN-g :11.2to 23,972(2.8,not detected,and 4.2);CXCL10:2.1to 5319(481,115,and 287);MIP-1b :3.3to 7233(99.7,371,and 174);MCP-1:4.8to 3600(403,560,and 828);CXCL9:48.2to 3700(1412,126,and 177);IL-2R a :13.4to 34,210(4319,9477,and 610);IL-8:2.4to 5278(15.3,14.5,and 14.6);IL-10:6.7to 13,874(8.5,5.4,and 0.7);MIP-1a :7.1to 13,778(57.6,57.3,and 48.1).o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mProfound antitumor clinical activity of CART19cellsThere were no significant toxicities observed during the4days after the infusion in any patient other than transient febrile reactions. However,all patients subsequently developed significant clinical and laboratory toxicities between days7and21after the first infusion. With the exception of B cell aplasia,these toxicities were short-term and reversible.Of the three patients treated to date,there are two complete responses and one partial response lasting greater than8months after CART19infusion according to standard criteria(22).Details of past medical history and response to therapy are described in Table1. The clinical course of UPN03has been described in detail(9).In brief,patient UPN02was treated with two cycles of bendamus-tine with rituximab,resulting in stable disease;he received a third dose of bendamustine as lymphodepleting chemotherapy before CART19 cell infusion.After CART19infusion,and coincident with the onset of high fevers,he had rapid clearance of the p53-deficient CLL cells from his PB(Fig.5A)and a partial reduction of adenopathy.He de-veloped fevers to40°C,rigors,and dyspnea requiring a24-hour hos-pitalization on day11after the first infusion and on the day of his second CART19cell boost.Fevers and constitutional symptoms per-sisted,and on day15,he had transient cardiac dysfunction;all symp-toms resolved after corticosteroid therapy was initiated on day18.His BM showed persistent extensive infiltration of CLL1month after therapy despite marked PB cytoreduction.He remained asymptomatic at the time of publication.Patient UPN01developed a febrile syndrome,with rigors and transient hypotension beginning10days after infusion.The fevers persisted for about2weeks and resolved;he has had no further consti-tutional symptoms.He achieved a rapid and complete response(Fig.5, B and C).Between1and6months after infusion,no circulating CLL cells were detected in the blood by deep sequencing(Table2).His BM at1,3,and6months after CART19cell infusions showed sustainedA1.40.67.890.223.58.02939.5CCR7CD28CD127CCR7CD28CD127C0.999.187.97.10.34.65.165.726.72.60.746.551.71.265.814.34.1715.71.81236.949.49.317.644.129CD4Day 169CCR7CD28CD127CCR7CD28CD127CD45RACD27CD25CD45RACD27CD25CD45RACD27CD25CD45RACD2744.017.34.234.4CD57CD5731.932.111.424.648.218.55.128.2CD57CD57PD-1PD-1PD-1PD-150.111.01127.927.841.716.613.874.719.81.73.8CD57CD57CD57CD5735.933.610.220.352.738.94.63.936.09.814.239.974.9230.61.5CD57CD57CD57CD57CD27CD28CD27CD28CD27CD28CD27CD28CD2565.00.60.433.719.959.411.39.231.551.66.610.030.147.910.811.12.459.635.42.639.59.28.842.5CCR7CD28CD127CD57CD57CD5779.516.70.90.36.833.234.925.110.928.041.319.82542.926.16.014.244.524.516.93.7 2.025.468.875.623.70.20.56.040.339.314.316.328.831.922.937.242.318.12.414.338.222.225.36.0 4.530.359.296.6 3.414.67.41464.15.115.760.418.89.411.464.614.60.60.12.596.818.473.03.05.697.10.70.062.111.2 3.111.873.95.58.052.833.77.8 6.454.231.635.559.21.33.927.264.316.779.20.200.898.97.500.891.73.60.10.395.97.60.40.191.90.10.213.885.90.20.312.387.2C00.284.715.100.969.329.80.10.21188.60.20.56.692.74.00.21.194.73.396.6Fig.4.Prolonged surface CAR19expressionand establishment of functional memoryCART19cells in vivo.(A and B)T cell immuno-phenotyping of CD4+(A)and CD8+(B)T cellsubsets.Frozen peripheral blood(PB)samplesfrom UPN03obtained at days56and169afterT cell infusion were subjected to multipara-metric immunophenotyping for expression ofmarkers of T cell memory,activation,and ex-haustion;data are displayed after biexponentialtransformation for objective visualization ofevents.(C)Functional competence of persistingCAR cells.Frozen PB samples from UPN03ob-tained at days56and169after T cell infusion were evaluated directly ex vivo for the ability to recognize CD19-expressing target cells using CD107 degranulation assays.Presented data are for the CD8+gated population.The gating strategies for these figures are presented in fig.S2.onFebruary2,212stm.sciencemag.orgDownloadedfrom。
2007_UPLC仪器操作_MS
冲洗进样针(Wash Needle)
冲洗进样针是一项可选程序，它是让强洗针液和/或弱洗针液 通过进样针和进样口部分 清洗进样针可以去掉进样针、刺穿针和进样口的外部和内部 的残留 可以通过进行冲洗针的操作来探知通过废液管的流路是否通 畅,以及确证洗针系统是否被灌注并运行正常 要求: 不要使用缓冲盐作为洗针液. 提示: 灌注系统已包含冲洗进样针, 所以只要做了灌注系统,就 可以省略此步操作
©2006 Waters Corporation
Prime Seal wash的步骤
将Seal wash出口端从废液盘上摘下,连接到注射器上 选择 Control>Prime Sealwash,点击之
©2006 Waters Corporation
Prime Seal wash的步骤
点击后,可以听到Seal wash 阀的开关声.此时: 慢慢抽注射器,直到有液体流出,然后将出口管再 接回到废液盘上 或者取下吸滤头,用吸满清洗液的注射器,从入口 端注入清洗液,直至出口管有液体流出 点击 可停止Prime过程
在下列情况下需要Prime二元溶剂管理器
•启用新的二元溶剂管理器或新的系统 •停机4小时后再次开机 •更换流动相
©2006 Waters Corporation
BSM 湿灌注的步骤
在此界面下,选择 Control>Prime A/B Solvents.
©2006 Waters Corporation
©2006 Waters Corporation
开机前的准备工作
UPLC对流动相的要求 UPLC对洗针液的要求
–有机溶剂要求用色谱纯(进口名牌最好) –水或缓冲盐流动相要用超纯水(18.2MΩ),当天制备 –强洗针液用甲醇/水或乙腈/水(有机相的比例高 于梯度洗脱的强溶剂的比例) –弱洗针液用纯水或含少量甲醇或乙腈的水溶 液(有机相的比例低于梯度洗脱的起始比例) –超纯水含5%-10%甲醇
布拉班德黏度仪 说明书
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stemcells英语解释
"Stem cells" refers to a special type of undifferentiated cell that has the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. There are two main types of stem cells: embryonic stem cells and adult stem cells. Embryonic stem cells can be found in embryos and have the potential to become any type of cell in the body, while adult stem cells are found in various tissues and are more limited in the types of cells they can become. Stem cells have the potential to revolutionize medicine and are the subject of much research and debate due to their unique properties and potential applications in treating a wide range of diseases and injuries.。
自噬通量染色rep-lc3步骤
自噬通量染色(rep-lc3)是一种用于研究细胞自噬的技术。
自噬是细胞内一种重要的代谢过程，通过这一过程，细胞可以清除老化或异常的细胞器，并回收其中的有用物质。
自噬通量染色(rep-lc3)可以帮助研究人员观察和分析自噬的过程，从而更好地了解细胞的代谢机制和相关疾病的发生机制。
下面将介绍自噬通量染色(rep-lc3)的步骤。
1. 细胞培养和处理在进行自噬通量染色(rep-lc3)之前，首先需要培养细胞并进行相应的处理。
这包括选择适当的细胞系，并将其培养在含有适当营养物质的培养基中。
在实验前，还需要根据具体实验设计对细胞进行处理，比如添加特定的药物或刺激条件来诱导自噬的发生。
这一步骤的关键在于保证细胞的健康和活力，以保证后续实验的准确性和可靠性。
2. 固定和染色接下来，将处理后的细胞进行固定和染色。
通常，这包括使用适当的固定剂（如甲醛或乙醇）来使细胞保持在原始状态，并使用LC3抗体来染色。
LC3是细胞中自噬小体的标志物，通过对其染色可以观察到自噬小体的形成和分布情况。
在这一步骤中，需要注意固定和染色的条件，以确保获得清晰而可靠的染色结果。
3. 显微镜观察和图像获取经过固定和染色后的细胞样品可以使用荧光显微镜进行观察。
通过观察细胞中LC3的染色情况，可以获得关于自噬活性和自噬小体形成的信息。
可以使用数字图像采集系统来获取高质量的显微图像，以备后续的数据分析和比较。
4. 数据分析和解释根据观察到的染色结果进行数据分析和解释。
通过对自噬小体的数量和分布进行定量分析，可以获得关于自噬通量的信息。
这些数据可以用来比较不同实验条件下自噬活性的变化，或者研究不同细胞类型之间的差异。
在解释数据时，需要注意排除可能的干扰因素，并结合相关的文献和实验控制来得出可靠的结论。
自噬通量染色(rep-lc3)是一种用于研究细胞自噬的重要技术。
通过严谨的实验设计和严格的操作流程，可以获得准确和可靠的结果，从而更好地了解自噬的调控机制和相关疾病的发生机制。
EM638325TS-5_-5G
EtronTechEM638325Etron Technology, Inc.No. 6, Technology Rd. V, Science-Based Industrial Park, Hsinchu, Taiwan 30077, R.O.C TEL: (886)-3-5782345 FAX: (886)-3-57786712M x 32 Synchronous DRAM (SDRAM)Preliminary (Rev 1.4 October/2005)Features• Clock rate: 200/183/166/143/125/100 MHz • Fully synchronous operation • Internal pipelined architecture• Four internal banks (512K x 32bit x 4bank) • Programmable Mode - CAS# Latency: 2 or 3- Burst Length: 1, 2, 4, 8, or full page - Burst Type: interleaved or linear burst - Burst-Read-Single-Write • Burst stop function• Individual byte controlled by DQM0-3 • Auto Refresh and Self Refresh • 4096 refresh cycles/64ms• Single +3.3V ± 0.3V power supply • Interface: LVTTL• Package: 400 x 875 mil, 86 Pin TSOP II, 0.50mm pin pitch• Lead Free Package availableOrdering InformationPart NumberLeaded / Lead Free Package FrequencyPackageEM638325TS-5/-5G 200MHz TSOP II EM638325TS-5.5/-5.5G 183MHz TSOP II EM638325TS-6/-6G 166MHz TSOP II EM638325TS-7/-7G 143MHz TSOP II EM638325TS-8/-8G 125MHz TSOP II EM638325TS-10/-10G100MHzTSOP IIPin Assignment (Top View)VDD DQ0VDDQ DQ1DQ2VSSQ DQ3DQ4VDDQ DQ5DQ6VSSQ DQ7NC VDD DQM0/WE /CAS /RAS /CS NC BS0BS1A10/APA0A1A2DQM2VDD NC DQ16VSSQ DQ17DQ18VDDQ DQ19DQ20VSSQ DQ21DQ22VDDQ DQ23VDDOverviewThe EM638325 SDRAM is a high-speed CMOS synchronous DRAM containing 64 Mbits. It is internally configured as a quad 512K x 32 DRAM with a synchronous interface (all signals are registered on the positive edge of the clock signal, CLK). Each of the 512K x 32 bit banks is organized as 2048 rows by 256 columns by 32 bits. Read and write accesses to the SDRAM are burst oriented; accesses start at a selected location and continue for a programmed number of locations in a programmed sequence. Accesses begin with the registration of a BankActivate command which is then followed by a Read or Write command.The EM638325 provides for programmable Read or Write burst lengths of 1, 2, 4, 8, or full page, with a burst termination option. An auto precharge function may be enabled to provide a self-timed row precharge that is initiated at the end of the burst sequence. The refresh functions, either Auto or Self Refresh are easy to use.By having a programmable mode register, the system can choose the most suitable modes to maximize its performance. These devices are well suited for applications requiring high memory bandwidth.Block DiagramA AB B D Q M 0~3APin DescriptionsTable 1. Pin Details of EM638325Symbol Type DescriptionCLK Input Clock:CLK is driven by the system clock. All SDRAM input signals are sampled on the positive edge of CLK. CLK also increments the internal burst counter and controls theoutput registers.CKE Input Clock Enable:CKE activates(HIGH) and deactivates(LOW) the CLK signal. If CKE goes low synchronously with clock(set-up and hold time same as other inputs), the internal clockis suspended from the next clock cycle and the state of output and burst address is frozenas long as the CKE remains low. When all banks are in the idle state, deactivating the clockcontrols the entry to the Power Down and Self Refresh modes. CKE is synchronous exceptafter the device enters Power Down and Self Refresh modes, where CKE becomesasynchronous until exiting the same mode. The input buffers, including CLK, are disabledduring Power Down and Self Refresh modes, providing low standby power.BS0, BS1 Input Bank Select:BS0 and BS1 defines to which bank the BankActivate, Read, Write, or BankPrecharge command is being applied. BS is also used to program the 11th bit of the Mode and Special Mode registers.A0-A10 Input Address Inputs: A0-A10 are sampled during the BankActivate command (row address A0-A10) and Read/Write command (column address A0-A7 with A10 defining Auto Precharge)to select one location out of the 256K available in the respective bank. During a Prechargecommand, A10 is sampled to determine if all banks are to be precharged (A10 = HIGH).The address inputs also provide the op-code during a Mode Register Set or Special ModeRegister Set command.CS# Input Chip Select:CS# enables (sampled LOW) and disables (sampled HIGH) the command decoder. All commands are masked when CS# is sampled HIGH. CS# provides for externalbank selection on systems with multiple banks. It is considered part of the command code. RAS# Input Row Address Strobe: The RAS# signal defines the operation commands in conjunction with the CAS# and WE# signals and is latched at the positive edges of CLK. When RAS#and CS# are asserted "LOW" and CAS# is asserted "HIGH," either the BankActivatecommand or the Precharge command is selected by the WE# signal. When the WE# isasserted "HIGH," the BankActivate command is selected and the bank designated by BS isturned on to the active state. When the WE# is asserted "LOW," the Precharge command isselected and the bank designated by BS is switched to the idle state after the prechargeoperation.CAS#Input Column Address Strobe:The CAS# signal defines the operation commands in conjunction with the RAS# and WE# signals and is latched at the positive edges of CLK.When RAS# is held "HIGH" and CS# is asserted "LOW," the column access is started byasserting CAS# "LOW." Then, the Read or Write command is selected by asserting WE#"LOW" or "HIGH."WE# Input Write Enable: The WE# signal defines the operation commands in conjunction with the RAS# and CAS# signals and is latched at the positive edges of CLK. The WE# input isused to select the BankActivate or Precharge command and Read or Write command.DQM0 - DQM3 Input Data Input/Output Mask: DQM0-DQM3 are byte specific, nonpersistent I/O buffer controls.The I/O buffers are placed in a high-z state when DQM is sampled HIGH. Input data is masked when DQM is sampled HIGH during a write cycle. Output data is masked (two-clock latency) when DQM is sampled HIGH during a read cycle. DQM3 masks DQ31-DQ24, DQM2 masks DQ23-DQ16, DQM1 masks DQ15-DQ8, and DQM0 masks DQ7-DQ0.DQ0-DQ31 Input/OutputData I/O: The DQ0-31 input and output data are synchronized with the positive edges of CLK. The I/Os are byte-maskable during Reads and Writes.NC - No Connect: These pins should be left unconnected.V DDQ Supply DQ Power: Provide isolated power to DQs for improved noise immunity. V SSQ Supply DQ Ground: Provide isolated ground to DQs for improved noise immunity. V DD Supply Power Supply: +3.3V±0.3VV SS Supply GroundOperation ModeFully synchronous operations are performed to latch the commands at the positive edges of CLK. Table 2 shows the truth table for the operation commands.Table 2. Truth Table (Note (1), (2) )CommandState CKE n-1 CKE n DQM (6) BS 0,1 A 10 A 9-0 CS# RAS# CAS# WE# BankActivate Idle (3) H X X V Row addressL L H H BankPrecharge Any H X X V L X L L H L PrechargeAll Any H X X X H XL L H L WriteActive (3) H X X V L L H L L Write and AutoPrecharge Active (3) H X X V H Column address (A0 ~ A7) L H L L ReadActive (3) H X X V L L H L H Read and Autoprecharge Active (3) H X X VH Column address (A0 ~ A7)L H L H Mode Register Set Idle H X X OP codeLL L L No-Operation Any H X X X X X L H H H Burst Stop Active (4) H X X X X X L H H L Device Deselect Any H X X X X X H X X X AutoRefresh Idle H H X X X X L L L H SelfRefresh Entry Idle H L X X X X L L L H SelfRefresh Exit IdleL H X X X X H X X X(SelfRefresh)L H H H Clock Suspend Mode Entry Active H L X X X X X X X X Power Down Mode Entry Any (5) H L X X X X H X X XL H H H Clock Suspend Mode Exit Active L H X X X X X X X X Power Down Mode Exit AnyL H X X X X H X X X(PowerDown)L H H H Data Write/Output Enable ActiveHXLX XXXXXX Data Mask/Output DisableActive H X H X X X X X XX2. CKE n signal is input level when commands are provided.CKE n-1 signal is input level one clock cycle before the commands are provided. 3. These are states of bank designated by BS signal.4. Device state is 1, 2, 4, 8, and full page burst operation.5. Power Down Mode can not enter in the burst operation.When this command is asserted in the burst cycle, device state is clock suspend mode. 6. DQM0-3Commands1 BankActivate(RAS# = "L", CAS# = "H", WE# = "H", BS = Bank, A0-A10 = Row Address)The BankActivate command activates the idle bank designated by the BS0,1 (Bank Select) signal. By latching the row address on A0 to A10 at the time of this command, the selected row access is initiated. The read or write operation in the same bank can occur after a time delay of t RCD (min.) from the time of bank activation. A subsequent BankActivate command to a different row in the same bank can only be issued after the previous active row has been precharged (refer to the following figure). The minimum time interval between successive BankActivate commands to the same bank is defined by t RC (min.). The SDRAM has four internal banks on the same chip and shares part of the internal circuitry to reduce chip area; therefore it restricts the back-to-back activation of the four banks. t RRD (min.) specifies the minimum time required between activating different banks. After this command is used, the Write command and the Block Write command perform the no mask write operation.CLK ADDRESST0T 1T2T3Tn+3Tn+4T n+5Tn+6COM MANDBegin: "H" or "L"BankActivate Command Cycle (Burst Length = n, CAS# Latency = 3)2 BankPrecharge command(RAS# = "L", CAS# = "H", WE# = "L", BS = Bank, A10 = "L", A0-A9 = Don't care)The BankPrecharge command precharges the bank disignated by BS0,1 signal. The precharged bank is switched from the active state to the idle state. This command can be asserted anytime after t RAS (min.) is satisfied from the BankActivate command in the desired bank. The maximum time any bank can be active is specified by t RAS (max.). Therefore, the precharge function must be performed in any active bank within t RAS (max.). At the end of precharge, the precharged bank is still in the idle state and is ready to be activated again.3 PrechargeAll command(RAS# = "L", CAS# = "H", WE# = "L", BS = Don ’t care, A10 = "H", A0-A9 = Don't care) The PrechargeAll command precharges all the four banks simultaneously and can be issued even if all banks are not in the active state. All banks are then switched to the idle state.4 Read command(RAS# = "H", CAS# = "L", WE# = "H", BS = Bank, A10 = "L", A0-A7 = Column Address)The Read command is used to read a burst of data on consecutive clock cycles from an active row in an active bank. The bank must be active for at least t RCD (min.) before the Read command is issued. During read bursts, the valid data-out element from the starting column address will be available following the CAS# latency after the issue of the Read command. Each subsequent data-out element will be valid by the next positive clock edge (refer to the following figure). The DQs go into high-impedance at the end of the burst unless other command is initiated. The burst length, burst sequence, and CAS# latency are determined by the mode register which is already programmed. A full-page burst will continue until terminated (at the end of the page it will wrap to column 0 and continue).CLK COMMANDCAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'sBurst Read Operation(Burst Length = 4, CAS# Latency = 2, 3)The read data appears on the DQs subject to the values on the DQM inputs two clocks earlier (i.e. DQM latency is two clocks for output buffers). A read burst without the auto precharge function may be interrupted by a subsequent Read or Write command to the same bank or the other active bank before the end of the burst length. It may be interrupted by a BankPrecharge/ PrechargeAll command to the same bank too. The interrupt coming from the Read command can occur on any clock cycle following a previous Read command (refer to the following figure).CLK COMMANDCAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'sT0T 1T2T3T4T5T6T7T8Read Interrupted by a Read (Burst Length = 4, CAS# Latency = 2, 3)The DQM inputs are used to avoid I/O contention on the DQ pins when the interrupt comes from a Write command. The DQMs must be asserted (HIGH) at least two clocks prior to the Write command to suppress data-out on the DQ pins. To guarantee the DQ pins against I/O contention, a single cycle with high-impedance on the DQ pins must occur between the last read data and the Write command (refer to the following three figures). If the data output of the burst read occurs at the second clock of the burst write, the DQMs must be asserted (HIGH) at least one clock prior to the Write command to avoid internal bus contention.CLK DQMCOM MAND DQ'sRead to Write Interval (Burst Length ≧ 4, CAS# Latency = 3)CLK DQMCOMMAND T0T 1T2T3T4T5T6T7T8CAS# latency=2t CK2, DQ'sRead to Write Interval (Burst Length ≥ 4, CAS# Latency = 2)CLK DQMCOMMAND T0T 1T2T3T4T5T6T7T8CAS# latency=2, DQ'sRead to Write Interval (Burst Length ≥ 4, CAS# Latency = 2)A read burst without the auto precharge function may be interrupted by a BankPrecharge/ PrechargeAll command to the same bank. The following figure shows the optimum time that BankPrecharge/ PrechargeAll command is issued in different CAS# latency.CLKCOMMANDCAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'sADDRESSRead to Precharge (CAS# Latency = 2, 3)5 Read and AutoPrecharge command(RAS# = "H", CAS# = "L", WE# = "H", BS = Bank, A10 = "H", A0-A7 = Column Address)The Read and AutoPrecharge command automatically performs the precharge operation after the read operation. Once this command is given, any subsequent command cannot occur within a time delay of {t RP (min.) + burst length}. At full-page burst, only the read operation is performed in this command and the auto precharge function is ignored.6 Write command(RAS# = "H", CAS# = "L", WE# = "L", BS = Bank, A10 = "L", A0-A7 = Column Address)The Write command is used to write a burst of data on consecutive clock cycles from an active row in an active bank. The bank must be active for at least t RCD (min.) before the Write command is issued. During write bursts, the first valid data-in element will be registered coincident with the Write command. Subsequent data elements will be registered on each successive positive clock edge (refer to the following figure). The DQs remain with high-impedance at the end of the burst unless another command is initiated. The burst length and burst sequence are determined by the mode register, which is already programmed. A full-page burst will continue until terminated (at the end of the page it will wrap to column 0 and continue).CLKCOM MAND DQ0 - DQ3are registered on the same clock edge.Burst Write Operation (Burst Length = 4, CAS# Latency = 1, 2, 3)A write burst without the AutoPrecharge function may be interrupted by a subsequent Write, BankPrecharge/PrechargeAll, or Read command before the end of the burst length. An interrupt coming from Write command can occur on any clock cycle following the previous Write command (refer to the following figure).CLK COMMANDDQ'sWrite Interrupted by a Write (Burst Length = 4, CAS# Latency = 1, 2, 3)The Read command that interrupts a write burst without auto precharge function should be issued one cycle after the clock edge in which the last data-in element is registered. In order to avoid data contention, input data must be removed from the DQs at least one clock cycle before the first read data appears on the outputs (refer to the following figure). Once the Read command is registered, the data inputs will be ignored and writes will not be executed.CLKCOMMAND data contention.CAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'sWrite Interrupted by a Read (Burst Length = 4, CAS# Latency = 2, 3)The BankPrecharge/PrechargeAll command that interrupts a write burst without the auto precharge function should be issued m cycles after the clock edge in which the last data-in element is registered, where m equals t WR /t CK rounded up to the next whole number. In addition, the DQM signals must be used to mask input data, starting with the clock edge following the last data-in element and ending with the clock edge on which the BankPrecharge/PrechargeAll command is entered (refer to the following figure).CLKT0T 1T2T3T4T 5T6COMMANDDQMADDRESSDQ: don't careNote: The DQMs can remain low in this example if the length of the write burst is 1 or 2.Write to Precharge(RAS# = "H", CAS# = "L", WE# = "L", BS = Bank, A10 = "H", A0-A7 = Column Address)The Write and AutoPrecharge command performs the precharge operation automatically after the write operation. Once this command is given, any subsequent command can not occur within a time delay of {(burst length -1) + t WR + t RP (min.)}. At full-page burst, only the write operation is performed in this command and the auto precharge function is ignored.CLK COMMANDT0T 1T2T3T4T5T6T7T8CAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'st DAL = t WR + t RP*Begin AutoPrechargeBank can be reactivated at completion of t DALBurst Write with Auto-Precharge (Burst Length = 2, CAS# Latency = 2, 3)8 Mode Register Set command(RAS# = "L", CAS# = "L", WE# = "L", BS0,1 and A10-A0 = Register Data)The mode register stores the data for controlling the various operating modes of SDRAM. The Mode Register Set command programs the values of CAS# latency, Addressing Mode and Burst Length in the Mode register to make SDRAM useful for a variety of different applications. The default values of the Mode Register after power-up are undefined; therefore this command must be issued at the power-up sequence. The state of pins BS0,1 and A10~A0 in the same cycle is the data written to the mode register. One clock cycle is required to complete the write in the mode register (refer to the following figure). The contents of the mode register can be changed using the same command and the clock cycle requirements during operation as long as all banks are in the idle state.RAS#CLKCKECS#CAS#WE#ADDR.DQMDQSet CommandCommandMode Register Set Cycle (CAS# Latency = 2, 3)The mode register is divided into various fields depending on functionality. Address BS0,1 A10/AP A9 A8A7A6A5A4A3 A2A1A0Function RFU*RFU*WBLTest Mode CAS Latency BTBurst Length*Note: RFU (Reserved for future use) should stay “0” during MRS cycle. • Burst Length Field (A2~A0)This field specifies the data length of column access using the A2~A0 pins and selects the Burst Length to be 2, 4, 8, or full page.A2 A1 A0 Burst Length0 0 0 1 0 0 1 2 0 1 0 4 0 1 1 8 1 0 0 Reserved 1 0 1 Reserved 1 1 0 Reserved 1 11Full PageThe Burst Type can be one of two modes, Interleave Mode or Sequential Mode.A3 Burst Type0 Sequential1 Interleave--- Addressing Sequence of Sequential ModeAn internal column address is performed by increasing the address from the column address which is input to the device. The internal column address is varied by the Burst Length as shown in the following table. When the value of column address, (n + m), in the table is larger than 255, only the least significant 8 bits are effective.Data n 0 1 2 3 4 5 6 7 - 255 256 257 -n n+1 - Column Address n n+1 n+2 n+3 n+4 n+5 n+6 n+7 - N+2552 words:Burst Length 4 words:8 words:Full Page: Column address is repeated until terminated.--- Addressing Sequence of Interleave ModeA column access is started in the input column address and is performed by inverting the address bits in the sequence shown in the following table.Data n Column Address Burst Length Data 0 A7 A6 A5 A4 A3 A2 A1 A0Data 1 A7 A6 A5 A4 A3 A2 A1 A0# 4 wordsData 2 A7 A6 A5 A4 A3 A2 A1# A0Data 3 A7 A6 A5 A4 A3 A2 A1# A0# 8 words Data 4 A7 A6 A5 A4 A3 A2# A1 A0Data 5 A7 A6 A5 A4 A3 A2# A1 A0#Data 6 A7 A6 A5 A4 A3 A2# A1# A0Data 7 A7 A6 A5 A4 A3 A2# A1# A0#•CAS# Latency Field (A6~A4)This field specifies the number of clock cycles from the assertion of the Read command to the first read data. The minimum whole value of CAS# Latency depends on the frequency of CLK. The minimum whole value satisfying the following formula must be programmed into this field.t CAC(min) ≤ CAS# Latency X t CKA6 A5 A4 CAS# Latency0 0 0 Reserved0 0 1 Reserved0 1 0 2 clocks0 1 1 3 clocks1 X X ReservedThese two bits are used to enter the test mode and must be programmed to "00" in normal operation.A8 A7 Test Mode 0normal mode0 1 Vendor Use Only 1 X Vendor Use Only• Write Burst Length (A9)This bit is used to select the burst write length.A9 Write Burst Length0 Burst 1Single Bit9 No-Operation command(RAS# = "H", CAS# = "H", WE# = "H")The No-Operation command is used to perform a NOP to the SDRAM which is selected (CS#is Low). This prevents unwanted commands from being registered during idle or wait states.10 Burst Stop command (RAS# = "H", CAS# = "H", WE# = "L") The Burst Stop command is used to terminate either fixed-length or full-page bursts. Thiscommand is only effective in a read/write burst without the auto precharge function. The terminated read burst ends after a delay equal to the CAS# latency (refer to the following figure). The termination of a write burst is shown in the following figure.CLKCOMMAND CAS# latency=2t CK2, DQ's CAS# latency=3t CK3, DQ'sTermination of a Burst Read Operation (Burst Length ＞ 4, CAS# Latency = 2, 3)CLKCOMMAND DQ'sTermination of a Burst Write Operation (Burst Length = X, CAS# Latency = 1, 2, 3)11 Device Deselect command (CS# = "H")The Device Deselect command disables the command decoder so that the RAS#, CAS#, WE# and Address inputs are ignored, regardless of whether the CLK is enabled. This command is similar to the No Operation command.12 AutoRefresh command(RAS# = "L", CAS# = "L", WE# = "H",CKE = "H", BS0,1 = “Don‘t care, A0-A10 = Don't care) The AutoRefresh command is used during normal operation of the SDRAM and is analogous to CAS#-before-RAS# (CBR) Refresh in conventional DRAMs. This command is non-persistent, so it must be issued each time a refresh is required. The addressing is generated by the internal refresh controller. This makes the address bits a "don't care" during an AutoRefresh command. The internal refresh counter increments automatically on every auto refresh cycle to all of the rows. The refresh operation must be performed 4096 times within 64ms. The time required to complete the auto refresh operation is specified by t RC(min.). To provide the AutoRefresh command, all banks need to be in the idle state and the device must not be in power down mode (CKE is high in the previous cycle). This command must be followed by NOPs until the auto refresh operation is completed. The precharge time requirement, t RP(min), must be met before successive auto refresh operations are performed.13 SelfRefresh Entry command(RAS# = "L", CAS# = "L", WE# = "H", CKE = "L", A0-A10 = Don't care)The SelfRefresh is another refresh mode available in the SDRAM. It is the preferred refresh mode for data retention and low power operation. Once the SelfRefresh command is registered, all the inputs to the SDRAM become "don't care" with the exception of CKE, which must remain LOW.The refresh addressing and timing is internally generated to reduce power consumption. The SDRAM may remain in SelfRefresh mode for an indefinite period. The SelfRefresh mode is exited by restarting the external clock and then asserting HIGH on CKE (SelfRefresh Exit command).14 SelfRefresh Exit command(CKE = "H", CS# = "H" or CKE = "H", RAS# = "H", CAS# = "H", WE# = "H")This command is used to exit from the SelfRefresh mode. Once this command is registered, NOP or Device Deselect commands must be issued for t RC(min.) because time is required for the completion of any bank currently being internally refreshed. If auto refresh cycles in bursts are performed during normal operation, a burst of 4096 auto refresh cycles should be completed just prior to entering and just after exiting the SelfRefresh mode.15 Clock Suspend Mode Entry / PowerDown Mode Entry command (CKE = "L")When the SDRAM is operating the burst cycle, the internal CLK is suspended(masked) from the subsequent cycle by issuing this command (asserting CKE "LOW"). The device operation is held intact while CLK is suspended. On the other hand, when all banks are in the idle state, this command performs entry into the PowerDown mode. All input and output buffers (except the CKE buffer) are turned off in the PowerDown mode. The device may not remain in the Clock Suspend or PowerDown state longer than the refresh period (64ms) since the command does not perform any refresh operations.16 Clock Suspend Mode Exit / PowerDown Mode Exit commandWhen the internal CLK has been suspended, the operation of the internal CLK is reinitiated from the subsequent cycle by providing this command (asserting CKE "HIGH"). When the device is in the PowerDown mode, the device exits this mode and all disabled buffers are turned on to the active state. t PDE(min.) is required when the device exits from the PowerDown mode. Any subsequent commands can be issued after one clock cycle from the end of this command.17 Data Write / Output Enable, Data Mask / Output Disable command (DQM = "L", "H")During a write cycle, the DQM signal functions as a Data Mask and can control every word of the input data. During a read cycle, the DQM functions as the controller of output buffers. DQM is also used for device selection, byte selection and bus control in a memory system.Absolute Maximum RatingSymbol Item Leaded Package Lead Free Package Unit Note V IN, V OUT Input, Output Voltage -1~4.6 V 1 V DD, V DDQ Power Supply Voltage - 1~4.6 V 1 T OPR Operating Temperature 0~70 °C 1 T STG Storage Temperature - 55~150 °C 1 T SOLDER Soldering Temperature (10s) 240 260 °C 1 P D Power Dissipation 1 W 1I OUT Short Circuit Output Current 50 mA 1Recommended D.C. Operating Conditions (Ta = 0~70°C)Symbol Parameter Min. Typ. Max. Unit Note V DD Power Supply Voltage 3.0 3.3 3.6 V 2 V DDQ Power Supply Voltage(for I/O Buffer) 3.0 3.3 3.6 V 2 V IH LVTTL Input High Voltage 2.0 －V DDQ + 0.3 V 2 V IL LVTTL Input Low Voltage - 0.3 －0.8 V 2Capacitance (V DD = 3.3V, f = 1MHz, Ta = 25°C)Symbol Parameter Min. Max. UnitC I Input Capacitance － 4.5 pFC I/O Input/Output Capacitance － 6.5 pF Note: These parameters are periodically sampled and are not 100%。
CellSensstandard软件主要功能操作说明
CellSensstandard软件主要功能操作说明⼀、明场实时图像调节及拍照保存1.打开cellSens软件后，⾸先在摄像控制⼯具窗⼝中切换相机芯⽚为“彩⾊模式”，此时相机⾃动切换为彩⾊芯⽚进⾏明场图像采图；接下来点击“实时观察”对样品进⾏预览，随后在摄像控制⼯具窗⼝中将曝光模式调节为“⾃动”，最后将曝光模式调节到“⼿动”，通过增加或减少曝光时间来获得满意的观察效果；2.如果明场图像背景不够⼲净，可以在摄像控制⼯具窗⼝中执⾏⽩平衡操作，点击“⽩平衡”后框选预览图像中样品之外的背景区域，即可执⾏⽩平衡；3.根据实验需求，调节拍照的分辨率后点击“拍照”即可完成对当前预览图像的及时拍照，对该图像另存到指定⽂件夹，图像默认保存格式为“TIF”格式。
⼆、荧光实时图像调节及拍照保存1.打开软件后，⾸先在摄像控制⼯具窗⼝中切换相机芯⽚为“灰度模式”，此时相机⾃动切换为⿊⽩芯⽚进⾏荧光图像采图；接下来勾选⾼级荧光模式“SFL”；2.随后点击“实时观察”对荧光图像进⾏预览，并在软件上⽅的⼯具栏中选择合适的荧光观测模式（如DAPI、FITC），将曝光模式调节为“⾃动”，根据荧光表达的强弱，可以将曝光模式切换到“⾃动”，通过增加曝光时间来使得荧光表达更亮，反之亦然；若调节曝光时间超过1s，可以适当调⾼“增益”值(默认是1，可以调节到2)来使得更短的曝光时间达到相同的荧光亮度；*1、如果觉得荧光样品的背景不够⼲净，可以执⾏⿊平衡操作，点击“⿊平衡”后框选预览图像中荧光样品之外的背景区域，即可执⾏⿊平衡；*2、根据实验需求，调节拍照的分辨率后点击“拍照”即可完成对当前预览图像的及时拍照，对该图像另存到指定⽂件夹，图像默认保存格式为“TIF”格式多通道图像：⽤⿊⽩相机芯⽚⾃定义荧光观测模式所拍出的荧光图像，该图像在软件中已默认添加并使⽤了相应通道的伪彩⾊，此时图像模式为“多通道，16 位图像”。
三、多通道荧光⾃动叠加（CS-ST MA）1. 将⼯具窗⼝切换⾄“流程管理”⼯具窗⼝；2. 选定“⾃动处理”选项；3. 单击“多通道”按钮，按钮将显⽰为已点击状态，此时该按钮内部的充实浅黄⾊的背景；4. 单击添加通道按钮，上下⽂菜单随即被打开；该上下⽂菜单中将列出所有当前软件设置的观测模式（DAPI、FITC、CY3、CY5等荧光观测模式）；5. 选定⾸先要采集的⾊彩通道，例如蓝⾊“FUW”，随后单击第⼀个⾊彩通道旁边的⼩加号，激活当前蓝⾊通道，并按软件提⽰将滤⾊块转盘切换⾄1号位；6. 随后将⼯具窗⼝切换⾄“摄像控制”⼯具窗⼝，点击“实时观察”对荧光样品进⾏曝光时间及增益值进⾏调节，达到⼀个最佳的观察效果；然后切换⾄“流程管理”⼯具窗⼝，单击读取设置按钮，将最佳观察效果时的曝光时间及增益值读取到当前所激活的荧光通道；7.后⾯可以重复操作第4-6步，添加想要与当前已激活通道（例：蓝⾊通道）组合的其他荧光通道；8.在流程管理⼯具窗⼝中，单击开始按钮，随后软件中的提⽰依次切换不同的荧光滤⾊块，每次切换相机都会⾃动拍⼀张荧光图像，待指定多通道组合中的滤⾊块转盘切换完成后，软件⾃动将不同通道下拍摄的多张荧光图像进⾏荧光叠加，图像窗⼝中将显⽰所有通道的叠加荧光图像；9.将多通道荧光图像另存为指定⽂件夹，默认的⽂件保存格式为“TIF”格式，此为原图保存；10.如果需要将单个通道下的荧光图像保存，点击“平铺”按钮后，将⿏标放置在需要保存的单个荧光通道图像上，⿏标右键点击“抽取”，软件会将该通道的荧光图像单独抽取出来；此时点击“图像”>“印⼊信息”>并选择“是”之后再对该荧光图像进⾏保存，即可得到⼀张包含伪彩信息的荧光图像，图像格式建议为“TIF”格式。
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Cells in suspension are tagged with fluorescent markers specific for undifferentiated stem cell Labeled cells are sent under pressure through a small nozzle and pass through an electric field A cell generates a negative charge if it fluoresces and a positive charge if it does not.
STEM CELL
Four types EMBRYONIC STEM CELLS ADULT STEM CELLS PLACENTAL STEM CELLS (Cord Blood Stem Cells) AMNIOTIC FLUID STEM CELLS
Three general properties: they are capable of dividing themselves for long periods they are unspecialized; and they can give rise to specialized cell types.
7) Inherited Immune System Disorders • Ataxia-Telangiectasia • Kostmann Syndrome • Leukocyte Adhesion Deficiency • DiGeorge Syndrome • Bare Lymphocyte Syndrome • Omenn’s Syndrome • Severe Combined Immunodeficiency • SCID with Adenosine Deaminase Deficiency • Absence of T & B Cells SCID • Absence of T Cells, Normal B Cell SCID • Common Variable Immunodeficiency • Wiskott-Aldrich Syndrome • X-Linked Lymphoproliferative Disorder Other Inherited Disorders • Lesch-Nyhan Syndrome • Cartilage-Hair Hypoplasia • Glanzmann Thrombasthenia • Osteopetrosis • Adrenoleukodystrophy • Ceroid Lipofuscinosis • Congenital Erythropoietic Porphyria • Sandhoff Disease
Diseases that are treated by stem cells are:
1) Acute Leukemia • Acute Lymphoblast Leukemia (ALL) • Acute Myelogenous Leukemia (AML) • Acute Biphenotypic Leukemia • Acute Undifferentiated Leukemia 2) Chronic Leukemia • Chronic Myelogenous Leukemia (CML) • Chronic Lymphocytic Leukemia (CLL) • Juvenile Chronic Myelogenous Leukemia (JCML) • Juvenile Myelomonocytic Leukemia (JMML) Syndromes • Myelodysplastic Syndromes • Amyloidosis • Chronic Myelomonocytic Leukemia (CMML) • Refractory Anemia (RA) • Refractory Anemia with Excess Blasts (RAEB) • Refractory Anemia with Excess Blasts in Transformation • (RAEB-T) • Refractory Anemia with Ringed Sideroblasts (RARS)
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Capable of colonizing the germ line and giving rise to egg or sperm cells.
Clonogenic, that is a single ES cell can give rise to a colony of genetically identical cells or clones, which have the same properties as the original cell. Expresses the transcription factor Oct-4, Can be induced to continue proliferating or to differentiate. Lacks the G1 checkpoint in the cell cycle. ES cells spend most of their time in the S phase of the cell cycle, during which they synthesize DNA. Unlike differentiated somatic cells, ES cells do not require any external stimulus to initiate DNA replication. Do not show X inactivation. In every somatic cell of a female mammal, one of the two X chromosomes becomes permanently inactivated. X inactivation does not occur in undifferentiated ES cells.
Stem Cell Markers
c-Kit Oct4 (ATGCAAAT) POU Family Protein CD34 CD38 Cd44 CD133 Nestin
SEPARATION OF STEM CELL
Stem cell
பைடு நூலகம்
FLUROSCENT ACTIVATED CELL SORTING Laser beam passes through one cell
9) Plasma Cell Disorders • Multiple Myeloma • Plasma Cell Leukemia • Waldenstrom’s Macroglobulinemia • Amyloidosis Abnormalities 1) Inherited Platelet Abnormalities Congenital Thrombocytopenia 2) Inherited Erythrocyte Abnormalities • Beta Thalassemia Major • Sickle Cell Disease • Blackfan-Diamond Anemia • Pure Red Cell Aplasia Other Malignancies • Ewing Sarcoma • Neuroblastoma • Renal Cell Carcinoma • Retinoblastoma • Brain tumor • Ovarian Cancer • Small Cell Lung Cancer • Testicular Cancer
Disorders 5) Inherited Metabolic Disorders 1) Stem Cell Disorders • Mucopolysaccharidoses (MPS) • Aplastic Anemia (Severe) • Hurler’s Syndrome (MPS-IH) • Fanconi Anemia • Scheie Syndrome (MPS-IS) • Paroxysmal Nocturnal Hemoglobinuria Hunter’s Syndrome (MPS-II) • • Congenital Cytopenia • Sanfilippo Syndrome (MPS-III) • Dyskeratosis Congenita • Morquio Syndrome (MPS-IV) 2) Myeloproliferative Disorders • Maroteaux-Lamy Syndrome (MPS-VI) • Acute Myelofibrosis • Sly Syndrome, Beta-Glucuronidase Deficiency • Agnogenic Myeloid Metaplasia • Adrenoleukodystrophy • Polycythemia Vera • Mucolipidosis II (I-cell Disease) • Essential Thrombocythemia • Krabbe Disease 3) Lymphoproliferative Disorders • Gaucher’s Disease • Non-Hodgkin’s Lymphoma • Niemann-Pick Disease • Hodgkin’s disease • Wolman Disease • Prolymphocytic Leukemia • Metachromatic Leukodystrophy 4) Phagocyte Disorders 6) Histiocytic Disorders • Chediak-Higashi Syndrome • Familial Erythrophagocytic Lymphohistiocytosis • Chronic Granulomatous Disease • Histiocytosis-X • Neutrophil Actin Deficiency • Hemophagocytosis • Langerhans’ Cell Histiocytosis • Reticular Dysgenesis