Fluorescein Diacetate_596-09-8_DataSheet_MedChemExpress
花粉生活力测定
实验九花粉生活力测定一、实验目的1．学习和了解植物花粉生活力测定方法及原理。
2．初步掌握稻、麦的花粉生活力测定方法。
二、内容说明农业常规育种工作中，为了进行人工辅助授粉和杂交授粉，尤其是杂交育种工作，为解决亲本花期不一致和远距离杂交的问题，通常需要早期采集和储藏花粉。
不同类群植物花粉在自然条件下的寿命，花粉的储藏条件，以及花粉生活力的测定有所区别，这一是由花粉本身的特征决定的，二是由贮藏条件决定的。
一般来说，禾谷类作物花粉的寿命较短，自花授粉植物花粉的寿命尤其短，如小麦在花药开裂后30min，花粉即由鲜黄色变为深黄色，此时己有大量花粉丧失活力。
在许多特殊条件下，花粉生活力的差异对于研究花粉-柱头的相互作用，作物改良与育种操作，基因库的保持，不亲和性与受精关系，生理调节对花粉萌发的影响和基因流等，均有非常重要的实践意义。
因此，对于外地采集来的花粉的短暂贮藏、花粉的生物学研究、自交或远缘杂交分析结实率或不结实原因、鉴定雄性不育系时，花粉生活力的测定显得很重要。
花粉生活力有很多表述法，为了方便起见，现将各种表述方法列表如下(表9-1)，以供参考。
表9-1 花粉生活力的各种表述法花粉生活力的测定方法较多，通常可分为萌发测定和不萌发测定两大类，常用的有：（1）形态鉴定法是一种简便的鉴定新采集花粉的方法。
可通过直接观察花粉在形态上有明显差异来鉴定花粉有无生活力。
发育不正常的花粉，内含物不充实而空秕，形状也不规则，大小参差不齐。
而正常花粉内含物充实饱满，形状规则，大小整齐。
因内部含有较多淀粉粒而遇1%I-IK溶液呈深紫色反应，遇水易涨而破裂。
一般适用于不育系及远缘杂交后代花粉形态和育性的鉴定。
（2）染色鉴定法（FCR法）用不同的化学试剂如双乙酸荧光素(fluorescein diacetate)、联苯胺茶酚等快速鉴定花粉生活力。
双乙酸荧光素是一种荧光染料，其本身不产生荧光，无极性，可以自由地透过完整的原生质膜。
Fluorescence, Luminescence,
Fluorescence: “an effect in which a substance releases electromagnetic radiation while absorbing another form of energy, but ceases to emit radiation immediately upon cessation of the input energy”; “the light emission of a given wavelength by a substance that is activated by light of a different wavelength.” Academic Press Dictionary of
Theory of Fluorescence
Fluorescence Theory:
/ASRG/ HPLC/fluorescence.html
The origins of fluorescence in minerals:
uhaul/supergraphics/rocks/fluorescence.html
Measurement of Fluorescence
Fluorescence indicates effective absorbance & re-emission of electromagnetic radiation (often in the visible range, ~300 - 800 nm, but may involve other ranges, e.g., x - ray.
Pigments: flavins, stilbenes, tryptophan, tyrosine, purines..
荧光素激发发射波长
505
534
Di-4-ANEPPS
496
705
Di-8-ANEPPS (non-ratio)
488
498
605
713
DiA (4-Di-16-ASP)
456
591
Dichlorodihydrofluorescein Diacetate (DCFH)
505
BTC-5N - atio Dye, Zn2+
459/417
517/532
Ratio Dye, Zn2+
Calcein
494
517
Calcein Blue
373
440
Calcium Crimson?
588, 589
611, 615
Calcium Green
501, 506
531
Calcium Green-1 Ca2+ Dye
336
517
Dansyl fluoride
356
none
DAPI
359
461
Dapoxyl
403
580
Dapoxyl 2
374
574
Dapoxyl 3
373
574
DCFDA
504
529
DCFH (Dichlorodihydrofluorescein Diacetate)
505
535
DDAO
463
607
Berberine Sulphate
430
550
Beta Lactamase
409
447, 520
Flourence polarization(荧光偏振)
Polarization
Light is a harmonic electromagnetic wave. When considering its interaction with matter we can in most cases neglect the magnetic part. The plain in which the electric vector E oscillates defines the polarization of light.
Polarization in a fluorescence experiment
Z
excitation
X
I
I detection Y
The polarization state of fluorescence is described by: Polarization
Ill I P Ill I Ill I Ill I r IT Ill 2I
B
B
E
n E c
I E2
Natural light contains randomly all possible orientations of electric vector
Unpolarized (random) light
Polarizer
an optical component that selects from passing light only the component polarized in a given direction
hnexc hnexc
hnem
hnem > hnexc
Folin-Ciocalteu比色法测定黑莓多酚的含量
置于 ５ ｚ４ ０ ，０℃超声仪 中超声 提取 ４ ｉ， Ｈ ０ｍ ｎ 提取 完成
后 ， ０ｒ ｉ 离心 １ ｉ ，取上清液定容 至 ２０ｍ ， ４ ０ ｍ ｎ ０ ／ ０ｍ ｎ ０ Ｌ
在室温（０ Ｃ 、５３ 、０ ２ ） 、０４ ℃下 ， ｏ ２ 分别反应 １ 、 、 、２ 、 ５３ ６ １０ ０ ０ １０ｒ ｎ ８ ｉ 后在 ７ ５ ｍ下测定吸光度值 。 ａ ６ ｎ
没食子酸标准品、ｏ ｎ Ｃｏａｔ 试剂 （ ｏ Ｌ 、 Ｆｌ － ｉ ｌ ｕＳ ｉ ｃ ｅ ２ Ｉ ） ｍ Ｊ
碳 酸钠 、 乙醇为分析纯 ； Ｘ ２ ０ Ｅ 一 ０ Ａ型 电子天平 ： 慈溪 市 天东衡器 厂 ； Ｒ ８ ８ Ｈ ＬＰ 搅 拌机 ：飞利 浦 电子公 Ｈ ２ ３Ｐ ＩＩＳ 司 ；Ｕ １ １ Ｔ 一 ８０紫外可见分光光度计 ： 北京普析通 用仪器
ｇ ｌ ｃ ａ ｉ ｓｓａ ｄ ｒ ． ｈ ｐ ｉ ｍ ｏ ｄｔｏ ｓｗｅ ｅａ ｈ ｅ ｅ ． ｈ ｅ ｕ ｔ ｈ ｗｅ ｈ ｔｗｈ ｎ２． １ｍｏＪＬ ａ ｌ ｃｄ ａ ｔｎ ａ ｄ ｔ ｅ ｏ ｔ ｉ ｍｕ ｃ ｎ ｉ ｎ ｒ ｃ ｉｖ ｄ Ｔ ｅ ｒ ｓ ｌｓｓ ｏ ｄ ｔ ａ ｅ ５ ｍＬ ０． ｌ ｉ Ｆｏｉ — ｏ ａｔ ｕ ａ ｄ ２ ｍＬ ． ｌｎ Ｃｉｃ ｌ ｎ ｅ ７５％ ｓ ｄｕ ｃ ｒ ｏ ａ ｅｓ ｌ ｔ ｎ ｗｅ ｅａ ｄ ｄ ｔ ｈ ５ ｍＬ ｓ ｍｐｅ ｒ ａ ｔｎ ２ ｎ ｏ ｉｍ ａ ｂ ｎ ｔ ｏｕ ｉ ｒ ｄ ｅ ｏｔ ｅ０． ａ ｌ ｅ ｃｉ ｇ １ ０ ｍｉ ｏ
每周只需注射一次,3个月即可轻松减掉10斤肥肉能让你管住嘴的减肥神药真的来了 临床大发现
每周只需注射一次，3个月即可轻松减掉10斤肥肉。
能让你管住嘴的减肥神药真的来了临床大发现“管住嘴，迈开腿”简简单单六个字，就道出了减肥的真谛。
然而，面对那么多的美食诱惑，光这前三个字就足以让无数人的减肥大业半途而废了。
不过，好消息来了！最近，肥胖研究领域中的著名期刊《糖尿病，肥胖和代谢》杂志刊登的一项临床研究[1]显示，诺和诺德公司开发的索马鲁肽，可以抑制食欲，让你轻松“管住嘴”。
只需一周注射1次，连续注射12周后，就可减重10斤！而且，在这减轻的体重中，主要还是体内的脂肪组织，药物对除脂肪以外的去脂体重影响很小。
不光有效，还很安全！这项研究的通讯作者，来自英国利兹大学的John Blundell 教授表示，“索马鲁肽的作用是非常令人惊讶的，我们在12周内就观察到了其他减肥药物需要6个月才能达到的效果。
它减少了饥饿感和食欲，让患者能更好地控制饮食摄入。
”[2] John Blundell教授索马鲁肽（Semaglutide）本身是一款针对2型糖尿病的降糖药，主要成分为胰高血糖素样肽-1（GLP-1）类似物。
GLP-1是一种由小肠分泌的激素，在血液中葡萄糖水平升高时促进胰岛素的合成和分泌。
GLP-1进入人体后很容易被酶降解，天然的GLP-1半衰期仅有几分钟，所以，为了让它更长久的工作，研究人员会对它进行一些结构上的改造，在保留功能的同时不那么容易被酶降解。
这样得到的GLP-1类似物药物，比如大名鼎鼎的利拉鲁肽，可以将注射频率减缓到每天1~2次。
而索马鲁肽可以说是它们的“升级版”，在经过改造后，它的半衰期可延长至大约1周，因此注射一次的效果可以维持大约一周的时间[3]，对于患者来说更方便。
在不久前公布的全球大型III期临床试验中，索马鲁肽表现优秀，既能控制血糖，还可以保护心血管，这为它在上周赢得了FDA内分泌及代谢药物专家咨询委员会16:0的支持率，不出意外的话，索马鲁肽上市在即[4]。
不少分析人士预测它未来十年内的销售峰值将超百亿，成为治疗2型糖尿病中最好的降糖药。
尼古丁对6-羟多巴胺诱导PC12细胞凋亡的拮抗作用
２ 结果
细胞损伤的保护 ２ ． １ 尼古丁对 ６ Ｏ Ｈ Ｄ Ａ所致 Ｐ Ｃ １ ２ 作用 正常对照组 Ｐ 细胞生长旺盛， 加入 ５ · Ｃ １ ２ ０ ｍ ｏ ｌ μ － １ ， 用Ｍ Ｌ ６ Ｏ Ｈ Ｄ Ａ Ｔ Ｔ法检测细胞的存活率为（ ４ ５ ± ） 加入不同浓度的尼古丁预处理 ２ｈ 后， 加入 ５ ８ ％， ０ － １ · 。结果显示， 尼古丁对 ６ ｍ ｏ ｌ Ｌ ６ Ｏ Ｈ Ｄ Ａ作用 ２ ４ｈ μ 细胞损伤有明显的保护作用， 作用 Ｏ Ｈ Ｄ Ａ所致 Ｐ Ｃ １ ２ 随尼古丁的浓度而变化， 呈钟型量 效关系， · １ ０ ｍ ｏ ｌ μ
］ ８ 细胞培养及凋亡模型的建立［ １ ． ２ Ｐ Ｃ １ ２
大鼠 肾 上 腺 嗜 铬 细 胞 瘤 细 胞 株 Ｐ Ｃ １ ２细 胞 在 培养基（ 内含 ５ Ｒ Ｐ Ｍ１ ６ ４ ０ ％马 血 清、 ５ ％小 牛 血 清 和 中分裂增殖， 并保持嗜铬细胞瘤的特 ５ ％胎牛血清） ， 细胞即停止分裂增 性。当培养基中加入 Ｎ Ｇ Ｆ Ｐ Ｃ １ ２ 殖， 胞体增大并形成突起， 表现交感神经元的特征。
Ｔ ａ ｂ２ ． Ａ ｎ ｔ ａ ｇ ｏ ｎ ｉ ｓ ｔ ｉ ｃｅ ｆ ｆ ｅ ｃ ｔ ｏ ｆ ｎ ｉ ｃ ｏ ｔ ｉ ｎ ｅｏ ｎＰ Ｃ １ ２ｃ ｅ ｌ ｌ ａ ｐ ｏ ｐ ｔ ｏ ｓ ｉ ｓｉ ｎ ｄ ｕ ｃ ｅ ｄｂ ｙ６ Ｏ Ｈ Ｄ Ａｗ ｉ ｔ ｈｆ ｌ ｕ ｏ ｒ ｅ ｓ ｃ ｅ ｉ ｎｄ ｉ ａ ｃ ｅ ｔ ａ ｔ ｅ ｎ ｕ ｃ ｌ ｅ ａ ｒ ｓ ｔ ａ ｉ ｎ ｉ ｎ ｇ
－ １ · 后， 细胞密度明显 ５ ０ ｍ ｏ ｌ Ｌ ６ Ｏ Ｈ Ｄ Ａ处理细胞 ２ ４ｈ μ 减少， 核仁聚集、 胞体皱缩、 正常胞突网状连接消失，
Agilent 8890 5977C Series gas chromatograph mass s
Agilent 5977C GC/MSD SystemThe Agilent 8890/5977C Series gas chromatograph/mass selective detector (GC/MSD) builds on a tradition of leadership in GC and MS technology, with the world’s most competitive performance and productivity features.Agilent GC/MSD system featuresAgilent 5977C GC/MSD — the most sensitive and robust MSD provides:–Four EI source options including the revolutionary high-efficiency source (HES), which offers the industry’s lowest instrument detection limit (IDL) and bestcarrier gas applications.signal-to-noise ratio (S/N) and a HydroInert source for H2– A heated monolithic quartz gold quadrupole (heatable up to 200 °C) for rapid elimination of contamination to keep the analyzer clean.– A second-generation triple-axis detector (TAD) for eliminating neutral noise.–Scan speeds up to 20,000 u/sec (extractor ion source and HES).–An optional oil-free IDP-3 roughing pump: a cleaner, quieter, and greener alternative (for use with turbo molecular pump systems).10-Year value promiseSupport is guaranteed for 10 years from the date of purchase, or Agilent will provide credit for the residual value of the system toward a model upgrade.Installation checkout specifications Agilent verifies GC/MSD system performance at the customer site.IDL is a statistically based metricthat more accurately confirms system performance than an S/N measurement. Test specificationsare based on splitless injection intoan Agilent J&W HP-5ms Ultra Inert30 m × 0.25 mm, 0.25 μm column for helium and a 20 m × 0.18 mm, 0.18 μm column for HydroInert with hydrogen. IDL analyses use lab helium (hydrogen for HydroInert) with GC gas filters installed. See more about the IDL test at /Library/ technicaloverviews/Public/5990-8341EN.pdf* IDL was statistically derived at 99% confidence level from the area precision of eight sequential splitless injections of OFN (octafluoronaphthalene). Demonstration of IDL specifications require a compatible system configuration, including a liquid autosampler with a 5 μL syringe.–HES IDL was measured using 10 fg injection, 1 µL injection.–Other IDLs were measured using 100 fg, 1 µL injection.–A 30 m column was used for helium IDL checkout; a 20 m column was used for hydrogenIDL checkout.–Helium carrier gas for Installation Specifications of the HES, Extractor, and Stainless steel sources; hydrogen carrier gas for Installation Specification of the HydroInert source only.–Reference IDL specifications from the above table will be confirmed only when purchased as an additional service with a compatible new system (GC and MS) installation.Signal-to-noise (S/N) specificationsa S/N checkout is performed only if there is no compatible autosampler (which is required for IDL checkout). Helium carrier gas, manual injection using a 30 m × 0.25 mm,0.25 µm column and in scan mode. Hydrogen carrier gas, manual injection using 20 m × 0.18 mm, 0.18 µm column and in scan mode. When the autosampler (ALS) is present, these specifications are a reference of the performance. Reference S/N specifications from the above table will not be confirmed at installation or introduction for ALS equipped systems.b Standard scanning from 50 to 300 u at nominal 272.0 u ion.c 1 μL injection of 100 pg/μL benzophenone (BZP) standard, 80 to 230 u scan at nominal 183 u ion, using methane reagent gas.d 2 μL injection of 100 fg/μL OFN standard scanning from 50 to 300 u at nominal 272 u ion, using methane reagent gas.2a Only applicable with optional Accurate Mass software package. Scan mode only. Not verified during installation.b As scan rate increases, sensitivity will decrease, and resolution may degrade.c A high flow rate into a fixed ion source will cause a loss in sensitivity.d The heated quadrupole mass filter should not require maintenance, but if maintenance is required, it should be performed by an Agilent service engineer.34aInlet temperature should be cool enough to touch when performing maintenance.bA micro ion gauge is shipped standard for the CI system, and is available optionally for EI systems.DE67854286This information is subject to change without notice.© Agilent Technologies, Inc. 2022Printed in the USA, May 26, 20225994-4846EN。
世界卫生组织儿童标准处方集
WHO Model Formulary for ChildrenBased on the Second Model List of Essential Medicines for Children 2009世界卫生组织儿童标准处方集基于2009年儿童基本用药的第二个标准目录WHO Library Cataloguing-in-Publication Data:WHO model formulary for children 2010.Based on the second model list of essential medicines for children 2009.1.Essential drugs.2.Formularies.3.Pharmaceutical preparations.4.Child.5.Drug utilization. I.World Health Organization.ISBN 978 92 4 159932 0 (NLM classification: QV 55)世界卫生组织实验室出版数据目录：世界卫生组织儿童标准处方集基于2009年儿童基本用药的第二个标准处方集1．基本药物 2.处方一览表 3.药品制备 4儿童 5.药物ISBN 978 92 4 159932 0 (美国国立医学图书馆分类：QV55)World Health Organization 2010All rights reserved. Publications of the World Health Organization can be obtained fromWHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: ******************). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the aboveaddress(fax:+41227914806;e-mail:*******************).世界卫生组织2010版权所有。
荧光光谱法英文
荧光光谱法英文Fluorescence SpectroscopyFluorescence spectroscopy is a powerful analytical technique that has found widespread applications in various fields, including chemistry, biology, materials science, and environmental studies. This analytical method is based on the measurement of the emission of light by a substance that has been excited by the absorption of light or other forms of energy. The process of fluorescence involves the absorption of energy by molecules or atoms, followed by the subsequent emission of light at a longer wavelength than the absorbed light.The fundamental principle of fluorescence spectroscopy is that when a molecule or atom is exposed to light, it can absorb the energy of the incoming photons, causing electrons within the molecule or atom to be excited to higher energy levels. This excitation is a temporary state, and the electrons will eventually return to their ground state, releasing the excess energy in the form of a photon. The energy of the emitted photon is typically lower than the energy of the absorbed photon, resulting in a shift in the wavelength of the emitted light compared to the absorbed light. This wavelength shift is known as the Stokes shift, and it is a key characteristic offluorescence.The intensity and wavelength of the emitted light are influenced by various factors, such as the chemical structure of the fluorescent molecule, the solvent environment, temperature, and the presence of other compounds that can interact with the excited molecules. By analyzing the characteristics of the emitted light, researchers can gain valuable insights into the properties and behavior of the sample under investigation.Fluorescence spectroscopy has a wide range of applications in various fields. In chemistry, it is used for the identification and quantification of organic and inorganic compounds, as well as the study of reaction kinetics and molecular interactions. In biology, fluorescence spectroscopy is employed for the investigation of protein structure and dynamics, the detection and quantification of biomolecules, and the study of cellular processes. In materials science, this technique is used to characterize the properties of polymers, semiconductors, and nanomaterials, among others.One of the key advantages of fluorescence spectroscopy is its high sensitivity, which allows for the detection and quantification of analytes at very low concentrations. Additionally, the technique is non-invasive and can be performed in real-time, making it a valuable tool for in-situ and online monitoring applications. Furthermore, thedevelopment of advanced fluorescent probes and labeling techniques has expanded the versatility of fluorescence spectroscopy, enabling the visualization and tracking of specific molecules or cellular components in complex biological systems.Despite its many benefits, fluorescence spectroscopy also faces some limitations. The presence of interfering compounds, quenching effects, and the potential for photobleaching of the fluorescent molecules can challenge the reliability and accuracy of the measurements. Researchers are constantly working to address these challenges through the development of new instrumentation, data analysis methods, and sample preparation techniques.In conclusion, fluorescence spectroscopy is a powerful and versatile analytical tool that has made significant contributions to various scientific disciplines. As technology continues to advance, the applications of this technique are expected to expand further, providing researchers with new opportunities to gain a deeper understanding of the world around us.。
FDA 水解酶(Fluorescein diacetate,FDA)试剂盒使用说明
FDA水解酶（Fluorescein diacetate，FDA）试剂盒使用说明微量法注意：正式测定之前选择2-3个预期差异大的样本做预测定。
货号：BC0485规格：100T/48S产品内容：标准液：液体4mL×1瓶，4℃保存。
试剂一：液体40mL×1瓶，4℃保存。
试剂二：粉剂1瓶，-20℃避光保存，临用前加4mL试剂三溶解。
试剂三：液体40mL×1瓶，4℃保存。
产品说明：FDA水解反应能很好的反应土壤中微生物活性和土壤质量的变化以及生态系统中有机质的转化，是土壤质量研究中的重要生物学指标之一。
FDA是一种无色化合物，在介质中能被许多土壤酶所催化水解，并经脱水反应，产生酶解终产物荧光素，稳定不易被分解，并在490nm处有强吸收峰，通过检测490nm处的吸光值变化可计算得FDA水解酶活性。
自备实验用品及仪器：天平、低温离心机、可见分光光度计/酶标仪、微量石英比色皿/96孔板、恒温水浴锅。
操作步骤：一、样本处理称取风干过20目筛土壤约0.05g，备用。
二、测定操作表空白管标准管对照管测定管样本（g）0.050.05标准液（μL）40双蒸水（μL）40试剂一（μL）200200240200试剂二（μL）40充分混匀，30℃，震荡1h试剂三（mL）16016016016010000g，25℃，离心5min，取200μL上清于微量石英比色皿/96孔板中测定490nm处吸光值A，△A1=A标准管-A空白管，△A2=A测定管-A对照管注意：空白管和标准管只需测定一次。
三、酶活性计算公式a.用微量石英比色皿测定的计算公式如下酶活性定义：每克土壤每天产生1μmol荧光素的量为一个酶活力单位。
FDA活性（μmol/d/g）=（△A2÷△A1×C标准品）×V反总÷W=2.4×（△A2÷△A1）÷Wb.用96孔板测定的计算公式如下酶活性定义：每克土壤每天产生1μmol荧光素的量为一个酶活力单位。
荧光吸收光谱和发射光谱（6）
Fluorochrome Absorption and Emission SpectraHere we present the absorption and emission spectra of the fluorochromes BD Biosciences Pharmingen conjugates to monoclonal antibodies and other proteins. Four of these fluorochromes, fluorescein isothiocyanate (FITC), R-phycoerythrin (R-PE), BD Cy-Chrome™, and PerCP, can be used with single-laser flow cytometers equipped with an argon-ion laser emitting light at 488 nm for three-color flow cytometric analysis (Fig. 1).BD Biosciences Pharmingen also offers monoclonal antibodies and proteins conjugated to allophycocyanin (APC) and avidin conjugated to Texas Red™. Both of these dyes are useful in experiments where multi-color analysis is desired using flow cytometers with dual laser capabilities (Fig 1). APC can b e excited by a helium-neon (HeNe) laser emitting light at 633 nm, by a krypton laser emitting light at 647 nm, or by a dye laser which can be conveniently tuned to emit light in the 550-650 nm range. (In a dye laser, the lasing medium is a solution of fluo rescent dye excited by a pump laser, usually an ion laser.) Texas Red ™ , available conjugated to avidin, can be excited by an argon-krypton mixed-gas laser at 568 nm, or with a dye laser, where both APC and Texas Red ™ can be used simultaneously. Because many combinations of lasers, detectors, filters and fluorochromes are possible for multi-color analysis, proper precautions need to be taken (i.e.,bandpass filters, dichroic mirrors, longpass filters, etc.) by the operator to ensure each fluorochrome is being detected by only one detector (Fig. 1). In the following descriptions, we give our recommendations for the ideal instrument set-up for use with our reagents.(summarized in Table1) Allophycocyanin (APC) is an accessory photosynthetic pigment found in bluegreen algae. Its molecular weight is approximately 105 kDa. APC has 6 phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in R-PE. It has a 650-nm wavelength absorption maximum (Fig. 2) and a 660-nm fluorescence emission maximum (Fig. 2). Using a 660 ± 10 nm BP filter will give optimum detection for this fluorochrome. APC can be used in flow cytometers equipped with dual lasers for multi-color analysis (Fig. 2). It can be excited by laser light between 600-640 nm. For this, we recommend a He-Ne laser at 633 nm, or a tunable dye laser tund between 600-640 nm.BD Cy-Chrome™ is a tandem conjugate system, with an absorption maximum of approximately 650 nm (Fig. 2), which combines R-phycoerythrin and a cyanine dye (MW 1.5 kDa). When excited by 488-nm light, the excited fluorochrome (R-PE) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent maximum is approximately 670 nm (Fig. 2). Using a 650-nm longpass filter will give optimum detection for this fluorochrome. The efficiency of the light energy transfer between the two fluorochromes can be seen in Fig. 2F where less than 5% of the absorbed light is lost as fluorescence at 575 nm by R-PE. Compared to other fluorescence energy-transfer systems used in flow cytometry (e.g., RED613™ , EDC, PerCP), BD Cy-Chrome™ is a superior fluorochrome for third color analysis because of its high emission intensity and broad spectrum. As with our R-PE conjugates, an average of one BD Cy-Chrome™ molecule is coupled per antibody or protein. Because of its broad absorption range (Fig. 2), BD Cy-Chrome™ is not recommended for use with du al-laser flow cytometers where excitation by both lasers is possible.Fluorescein isothiocyanate (FITC) is a fluorochrome with a molecular weight of 389 daltons and an absorption maximum at 495 nm (Fig. 2). Its excitation by 488-nm light leads to a fluorescence emission maximum around 520 nm (Fig. 2). Using a 530 ± 15 nm bandpass (BP) filter will give optimum detection for this fluorochrome. The isothiocyanate derivative (FITC) is the most widely used form for conjugation to antibodies and proteins, but other derivatives are available. FITC has a high quantum yield (efficiency of energy transfer from absorption to emission fluorescence) and approximately half of the absorbed photons are emitted as fluorescent light. The number of FITC molecules per conjugate partner (antibody, Avidin, Streptavidin, etc.) is usually in the range of three to five molecules.R-phycoerythrin (R-PE) is an accessory photosynthetic pigment found in red algae. In vivo, it functions to transfer light energy to chlorophyll during photosynthesis. In vitro, it is a 240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule. The large number of fluorochromes per PE molecule makeR-phycoerythrin an ideal pigment for flow cytometry applications. Its absorption maximum is 564 nm (Fi g. 2). When excited by 488-nm light, its fluorescence emission maximum is approximately 575 nm (Fig. 2). For single-laser flow cytometer use, we recommend using a 585 ± 21 nm BP filter for optimal detection (Fig. 1). When performing multi-color analysis with a dual-laser system, a tighter window of detection is required to compensate for the other conjugates being used (e.g.,Texas Red ™ ). For this, we recommend using a 575 ± 13-nm BP filter (Fig.1). Our conjugation chemistry yields an average of one R-PE molecule per antibody orprotein. The emitted light is collected in the fluorescence-2 (FL2) channel.PE-Texas Red™ is a tandem conjugate system which combines R-PE and Texas Red™ and has an absorption maximum of approximately 564 nm. When excited by 488-nm light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the Texas Red™ molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 615 nm. Special care must be taken when using PE-Texas Red™ conjugates in conjunction with R-PE as there is considerable spectral overlap in the emission profiles of both fluorochromes.Peridinin chlorophyll protein (PerCP) is a component of the photosynthetic apparatus found in the dinoflagellate,Glenodinium . PerCP is a protein complex with a molecular weight of approximately 35 kDa. When excited by light at 488 nm from an argon-ion laser, PerCP has a excitation maximum around 490 nm, with an emission spectrum which peaks at 675 nm. The emitted light is collected in the fluorescence-3 (FL3) channel. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on stream-in-air flow cytometers.PerCP-Cy5.5 is a tandem conjugate system than combines PerCP with a cyanine d ye (Cy5.5™) and has an absorption maximum of approximately 490 nm. When excited by 488-nm light, the excited fluorochrome (PerCP) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 694 nm. Using a 650 nm longpass filter will give optimum detection for this fluorochrome. The emitted light is collected in the fluorescence-3 (FL3) channel. PerCP-Cy5.5 is recommended for use with stream-in-air flow cytometers. APC-Cy7is a tandem conjugate system that combines APC and a cyanine dye (Cy7™) and has an absorption maximum of approximately 650 nm. When excited by light from a dye or HeNe laser, the excited fluorochrome (APC) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is approximately 767 nm. It is recommended that a 750-nm longpass filter be used along with a red-sensitive detector such as the Hammatsu R3896 PMT for this fluorochrome. Special filters are required when using APC-Cy7ª in conjunction with APC. It is recommended that special precautions be taken with PharRed conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.Texas Red™ is a sulfonyl chloride derivative of sulforhodamine 101 with a molecular weight of 625 daltons. BD Biosciences Pharmingen offers Texas Red ™ , conjugated to avidin, as a useful second step for multi-color analysis. Because it em its in the long wavelengths of the deep red region (Fig 2), Texas Red ™ has little spectral overlap with FITC. When performing multi-color analysis involving both Texas Red™ and R-PE, BD Biosciences Pharmingen recommends excitation of Texas Red ™ using a d ual-laser flow cytometer equipped with a tunable dye laser to avoid "leaking" into the PE detector. If a krypton laser, emitting light at 568 nm, is used, the laser light will "leak" into the R-PE channel. Texas Red ™ can be used in conjunction with APC for multi-color analysis when both dyes are excited in the 595-605 nm range with a dye laser. Texas Red ™ has an absorption maximum of 596 nm (Fig. 2). Its emission maximum, when excited by 595-600-nm laser light, is 615 nm (Fig. 2). Using a 620 ± 10-nm bandpass filter will give optimum detection for this fluorochrome (Fig. 1).Comparative staining using a monoclonal antibody (RA3-6B2; anti-B220; Cat. No. 557390/553084**) conjugated to different fluorochromes and analyzed on either BD FACSVantage™ (upper panels) or BDFACSCalibur™ (lower panels). The numbers indicate the ratio of the median fluorescence intensity of positive cells to the negative cells (signal to noise ratio). These plots demonstrate how choices in A) fluorochrome-conjugates or B) instrumentation can affect the fluorescence intensity observed for a given population.A. The differences observed between individual fluorochrome-conjugates can be affected by the mAbconjugated. Thus while in the example above the PE-conjugate is brighter than the BD Cy-Chrome™ -conjugate, when analyzed on the BD FACSCalibur™ , for many mAbs the Cy-Chrome ™ -conjugate results in a brighter stain. Contact BD Biosciences Pharmingen Technical Services for more information on specific reagents.B. Similarly different flow cytometers utilize different lasers and different fluorescence filter sets whichcan result in differences in signal to noise ratios when using the same reagent. Note that PEreagents tend to be brighter when used on a BD FACSCalibur™ while APC reagents are brighter on a BD FACSVantage™. Note changes of the signal to noise ratio depending on fluorchrome and instrument used.Enlarge imageFigure 1."Top schematic." A single laser flow cytometer with five parameters of detection. Two detectors detect the light scatter, and three photo-multiplier tubes (PMTs) detect the fluorescent signals. The bandpass filters are set up for optimal detection with BD Biosciences Pharmingen's fluorochromes: FITC, PE, BD Cy-Chrome ™ and Becton Dickinson's PerCP."Bottom schematic." A dual laser flow cytometer with six parameters of detection. Two detectors detect the light scatter, and four PMTs detect the fluorescent signals. The bandpass filters are set up forop timal detection with BD Biosciences Pharmingen's fluorochromes FITC, PE, APC, and Texas Red ™ . The second (orange) laser light is emitted from a tunable dye head using rhodamine 6 G as the fluorescent dye for excitation. Forward light scatter (FSC), side scatter (SSC), FITC, and PE signals are all produced by the primary 488-nm argon-ion laser. APC and Texas Red ™ signals are produced by the second laser (dye head with a 488-nm argon-ion laser).Figure 2. Absorption spectra of Fluorochromes. Individual fluorochrome excitation spectra are found in gray and the corresponding emission spectra in black. Typical band pass filters are given for eachflu orochrome as used on a FACSVantage™ except for BD Cy-Chrome™ and PerCP which are shown for FACSCalibur™ configurations.TABLE 1. Comparison of individual fluorochromes with single and dual laser flow cytometry.*PerCP is highly sensitive to photobleaching and must be used with laser power <150mW++Can only be used with a dye laser#Not recommended (dull)$BD Cy-Chrome and APC cannot be simultaneously used on instruments lacking cross-beam compensation. References:Loken, M.R., 1990. Immunofluorescence Techniques in Flow Cytometry and Sorting, 2nd Ed., Wiley. pp341-353.Parks, D., L. Herzenberg, and L. Herzenberg. 1989. Flow cytometry and fluorescence-activated cell sorting. Fundamental Immunology, Second Edition. William Paul, Ed.Raven Press, Ltd, New York.Zola, H. 1995. Detection of cytokine receptors by flow cytometry. In Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds.) John Wiley and Sons, New York. Unit 6.21.Immunofluorescence and cell sorting. In Current Protocols in Immunology. (J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, W. Strober, eds) John Wiley and Sons, New York. Unit 5.1 - 5.6.5. Shapiro, H.M. 1988. Practical Flow Cytometry, 2nd Ed. Wiley-Liss, New York.。
OECD Test No. 460 Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irrit
OECD/OCDE460Adopted: 2 October 2012© OECD, (2012)You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD.OECD GUIDELINE FOR THE TESTING OF CHEMICALS Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe IrritantsINTRODUCTION1. The Fluorescein Leakage (FL) test method is an in vitro test method that can be used under certain circumstances and with specific limitations to classify chemicals (substances and mixtures) as ocular corrosives and severe irritants, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Category 1), the European Union (EU) Regulation on Classification, Labelling and Packaging of Substances and Mixtures (CLP) (Category 1), and the U.S. Environmental Protection Agency (EPA) (Category I) (1) (2) (3). For the purpose of this Test Guideline, severe irritants are defined as chemicals that cause tissue damage in the eye following test substance administration that is not reversible within 21 days or causes serious physical decay of vision, while ocular corrosives are chemicals that cause irreversible tissue damage to the eye. These chemicals are classified as UN GHS Category 1, EU CLP Category 1, or U.S. EPA Category I.2. While the FL test method is not considered valid as a complete replacement for the in vivo rabbit eye test, the FL is recommended for use as part of a tiered testing strategy for regulatory classification and labelling. Thus, the FL is recommended as an initial step within a Top-Down approach to identify ocular corrosives/severe irritants, specifically for limited types of chemicals (i.e. water soluble substances and mixtures) (4)(5).3. It is currently generally accepted that, in the foreseeable future, no single in vitro eye irritation test will be able to replace the in vivo eye test (TG 405 (6)) to predict across the full range of irritation for different chemical classes. However, strategic combinations of several alternative test methods within a (tiered) testing strategy may be able to replace the in vivo eye test (5). The Top-Down approach (5) is designed to be used when, based on existing information, a chemical is expected to have high irritancy potential.Based on the prediction model detailed in paragraph 35, the FL test method can identify Category 1; EU CLP Category 1; U.S. EPA Category I) without any further testing. The same is assumed for mixtures although mixtures were not used in the validation. Therefore, the FL test method may be used to determine the eye irritancy/corrosivity of chemicals, following the460OECD/OCDEsequential testing strategy of TG 405 (6). However, a chemical that is not predicted as ocular corrosive or severe irritant with the FL test method would need to be tested in one or more additional test methods (in vitro and/or in vivo) that are capable of accurately identifying i) chemicals that are in vitro false negative ocular corrosives/severe irritants in the FL (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I); ii) chemicals that are not classified for eye corrosion/irritation (UN GHS No Category; EU CLP No Category; U.S. EPA Category IV); and/or iii) chemicals that are moderate/mild eye irritants (UN GHS Categories 2A and 2B; EU CLP Category 2; U.S. EPA Categories II and III).5. The purpose of this Test Guideline is to describe the procedures used to evaluate the potential ocular corrosivity or severe irritancy of a test substance as measured by its ability to induce damage to an impermeable confluent epithelial monolayer. The integrity of trans-epithelial permeability is a major function of an epithelium such as that found in the conjunctiva and the cornea. Trans-epithelial permeability is controlled by various tight junctions. Increasing the permeability of the corneal epithelium in vivo has been shown to correlate with the level of inflammation and surface damage observed as eye irritation develops.6. In the FL test method, toxic effects after a short exposure time to the test substance are measured by an increase in permeability of sodium fluorescein through the epithelial monolayer of Madin-Darby Canine Kidney (MDCK) cells cultured on permeable inserts. The amount of fluorescein leakage that occurs is proportional to the chemical-induced damage to the tight junctions, desmosomal junctions and cell membranes, and can be used to estimate the ocular toxicity potential of a test substance. Annex I provides a diagram of MDCK cells grown on an insert membrane for the FL test method.7. Definitions are provided in Annex II.INITIAL CONSIDERATIONS AND LIMITATIONS8. This Test Guideline is based on the INVITTOX protocol No. 71 (7) that has been evaluated in an international validation study by the European Centre for the Validation of Alternative Methods (ECVAM) (8), in collaboration with the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM).9. The FL test method is not recommended for the identification of chemicals which should be classified as mild/moderate irritants or of chemicals which should not be classified for ocular irritation (substances and mixtures) (i.e. GHS Cat. 2A/2B, no category; EU CLP Cat. 2, no category; US EPA Cat. II/III/IV), as demonstrated by the validation study (4) (8).10. The test method is only applicable to water soluble chemicals (substances and mixtures). The ocular severe irritation potential of chemicals that are water soluble and/or where the toxic effect is not affected by dilution is generally predicted accurately using the FL test method (8). To categorise a chemical as water soluble, under experimental conditions, it should be soluble in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, Hanks’ Buffered Salt Solution (HBSS) at a concentration ≥ 250 mg/mL (one dose above the cut-off of 100 mg/mL). However, if the test substance is soluble below the concentration 100 mg/mL,2© OECD, (2012)OECD/OCDE 460 but already induces a FL induction of 20 % at that concentration (meaning FL20 < 100 mg/mL), it can still be classified as GHS Cat. 1 or EPA Cat. 1.11. The identified limitations for this test method exclude strong acids and bases, cell fixatives and highly volatile chemicals from the applicability domain. These chemicals have mechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponification or specific reactive chemistries. Other identified limitations for this method are based upon the results for the predictive capacity for coloured and viscous test substance (8). It is suggested that both types of chemicals are difficult to remove from the monolayer following the short exposure period and that predictivity of the test method could be improved if a higher number of washing steps was used. Solid chemicals suspended in liquid have the propensity to precipitate out and the final concentration to cells can be difficult to determine. When substances within these chemical and physical classes are excluded from the database, the accuracy of FL across the EU, EPA, and GHS classification systems is substantially improved (8).12. Based on the purpose of this test method (i.e. to identify ocular corrosives/severe irritants only), false negative rates (see Paragraph 13) are not critical since such substances would be subsequently tested with other adequately validated in vitro tests or in rabbits, depending on regulatory requirements, using a sequential testing strategy in a weight of evidence approach (6) (see also paragraphs 3 and 4).13. Other identified limitations of the FL test method are based on false negative and false positive rates. When used as an initial step within a Top-Down approach to identify water soluble ocular corrosive/severe irritant substances and mixtures (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I), the false positive rate for the FL test method ranged from 7% (7/103; UN GHS and EU CLP) to 9% (9/99; U.S. EPA) and the false negative rate ranged from 54% (15/28; U.S. EPA) to 56% (27/48; UN GHS and EU CLP) when compared to in vivo results. Chemical groups showing false positive and/or false negative results in the FL test method are not defined here.14. Certain technical limitations are specific to the MDCK cell culture. The tight junctions that block the passage of the sodium-fluorescein dye through the monolayer are increasingly compromised with increasing cell passage number. Incomplete formation of the tight junctions results in increased FL in the non-treated control. Therefore, a defined permissible maximal leakage in the non-treated controls is important (see paragraph 38: 0% leakage). As with all in vitro assays there is the potential for the cells to become transformed over time, thus it is vital that passage number ranges for the assays are stated.15. The current applicability domain might be increased in some cases, but only after analyzing an expanded data set of studied test substances, preferably acquired through testing (4). This Test Guideline will be updated accordingly as new information and data are considered.16. For any laboratory initially establishing this assay, the proficiency chemicals provided in Annex III should be used. Laboratories can use these chemicals to demonstrate their technical competence in performing the FL test method prior to submitting FL assay data for regulatory hazard classification purposes.PRINCIPLE OF THE TEST3© OECD, (2012)460OECD/OCDE17. The FL test method is a cytotoxicity and cell-function based in vitro assay that is performed on a confluent monolayer of MDCK CB997 tubular epithelial cells that are grown on semi-permeable inserts and model the non-proliferating state of the in vivo corneal epithelium. The MDCK cell line is well established and forms tight junctions and desmosomal junctions similar to those found on the apical side of conjunctival and corneal epithelia. Tight and desmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal epithelium. Loss of trans-epithelial impermeability, due to damaged tight junctions and desmosomal junctions, is one of the early events in chemical-induced ocular irritation.18. The test substance is applied to the confluent layer of cells grown on the apical side of the insert. A short 1 min exposure is routinely used to reflect the normal clearance rate in human exposures. An advantage of the short exposure period is that water-based substances and mixtures can be tested neat, if they can be easily removed after the exposure period. This allows more direct comparisons of the results with the chemical effects in humans. The test substance is then removed and the non-toxic, highly fluorescent sodium-fluorescein dye is added to the apical side of the monolayer for 30 minutes. The damage caused by the test substance to the tight junctions is determined by the amount of fluorescein which leaks through the cell layer within a defined period of time.19. The amount of sodium-fluorescein dye that passes through the monolayer and the insert membrane into a set volume of solution present in the well (to which the sodium-fluorescein dye leaks in) is determined by measuring spectrofluorometrically the fluorescein concentration in the well. The amount of fluorescein leakage (FL) is calculated with reference to fluoresence intensity (FI) readings from two controls: a blank control, and a maximum leakage control. The percentage of leakage and therefore amount of damage to the tight junctions is expressed, relative to these controls, for each of the set concentrations of the test substance. Then the FL20 (i.e. concentration that causes 20% FL relative to the value recorded for the untreated confluent monolayer and inserts without cells), is calculated. The FL20 (mg/mL) value is used in the prediction model for identification of ocular corrosives and severe irritants (see paragraph 35).20. Recovery is an important part of a test substance’s toxicity profile that is also assessed by the in vivo ocular irritation test. Preliminary analyses indicated that recovery data (up to 72 h following the chemical exposure) could potentially increase the predictive capacity of INVITTOX Protocol 71 but further evaluation is needed and would benefit from additional data, preferably acquired by further testing (7). This Test Guideline will be updated accordingly as new information and data are considered.PROCEDUREPreparation of the cellular monolayer21. The monolayer of MDCK CB997 cells is prepared using sub-confluent cells growing in cell culture flasks in DMEM/Nutrient Mix F12 (1x concentrate with L-glutamine, 15 mM HEPES, calcium (at a concentration of 1.0-1.8 mM) and 10% heat-inactivated FCS/FBS). Importantly, all media/solutions used throughout the FL assay should contain calcium at a concentration between 1.8 mM (200 mg/L) and 1.0 mM (111 mg/L) to ensure tight junction formation and integrity. Cell passage number range should be controlled to ensure even and4© OECD, (2012)OECD/OCDE 460 reproducible tight junctions formation. Preferably, the cells should be within the passage range 3-30 from thawing because cells within this passage range have similar functionality, which aids assay results to be reproducible.22. Prior to performing the FL test method, the cells are detached from the flask by trypsinisation, centrifuged and an appropriate amount of cells is seeded into the inserts placed in 24-well plates (see Annex I). Twelve mm diameter inserts with membrane of mixed cellulose esters, a thickness of 80-150 µm and a pore size of 0.45 µm, should be used to seed the cells. In the validation study, Millicell-HA 12 mm inserts were used. The properties of the insert and membrane type are important as these may affect cell growth and chemical binding. Certain types of chemicals may bind to the Millicell-HA insert membrane, which could affect the interpretation of results. Proficiency chemicals (see Annex III) should be used to demonstrate equivalency if other membranes are used.23. Chemical binding to the insert membrane is more common for cationic chemicals, such as benzalkonium chloride, which are attracted to the positively charged membrane (8). Chemical binding to the insert membrane may increase the chemical exposure period, leading to an over-estimation of the toxic potential of the chemical, but can also physically reduce the leakage of fluorescein through the insert by binding of the dye to the cationic chemical bound to the insert membrane, leading to an under-estimation of the toxic potential of the chemical. This can be readily monitored by exposing the membrane alone to the top concentration of the chemical tested and then adding sodium-fluorescein dye at the normal concentration for the standard time (no cell control). If binding of the sodium-fluorescein dye occurs, the insert membrane appears yellow after the test material has been washed-off. Thus, it is essential to know the binding properties of the test substance in order to be able to interpret the effect of the chemical on the cells.24. Cell seeding on inserts should produce a confluent monolayer at the time of chemical exposure. 1.6 x 105 cells should be added per insert (400 µL of a cell suspension with a density of 4 x 105 cells / mL). Under these conditions, a confluent monolayer is usually obtained after 96 hours in culture. Inserts should be examined visually prior to seeding, so as to ensure that any damages recorded at the visual control described at paragraph 30 is due to handling.25. The MDCK cell cultures should be kept in incubators in a humidified atmosphere, at 5% ± 1% CO2and 37 ± 1 ºC. The cells should be free of contamination by bacteria, viruses, mycoplasma and fungi.Application of the Test and Control Chemicals26. A fresh stock solution of test substance should be prepared for each experimental run and used within 30 minutes of preparation. Test substances should be prepared in calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS to avoid serum protein binding. Solubility of the chemical at 250 mg/mL in HBSS should be assessed prior to testing. If at this concentration the chemical forms a stable suspension or emulsion (i.e.maintains uniformity and does not settle or separate into more than one phase) over 30 minutes, HBSS can still be used as solvent. However, if the chemical is found to be insoluble in HBSS at this concentration, the use of other test methods instead of FL should be considered. The use of light mineral oil as a solvent, in cases where the chemical is found to be insoluble in HBSS, should be5© OECD, (2012)460OECD/OCDEconsidered with caution as there is not enough data available to conclude on the performance of the FL assay under such conditions.27. All chemicals to be tested are prepared in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS from the stock solution, at five fixed concentrations diluted on a weight per volume basis: 1, 25, 100, 250 mg/mL and a neat or a saturated solution. When testing a solid chemical, a very high concentration of 750 mg/mL should be included. This concentration of chemical may have to be applied on the cells using a positive displacement pipette. If the toxicity is found to be between 25 and 100 mg/mL, the following additional concentrations should be tested twice: 1, 25, 50, 75, 100 mg/mL. The FL20value should be derived from these concentrations provided the acceptance criteria were met.28. The test substances are applied to the confluent cell monolayers after removal of the cell culture medium and washing twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS. Previously, the filters have been visually checked for any pre-existing damages that could be falsely attributed to potential incompatibilities with test chemicals. At least three replicates should be used for each concentration of the test substance and for the controls in each run. After 1 min of exposure at room temperature, the test substance should be carefully removed by aspiration, the monolayer should be washed twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS, and the fluorescein leakage should be immediately measured. 29. Concurrent negative (NC) and positive controls (PC) should be used in each run to demonstrate that monolayer integrity (NC) and sensitivity of the cells (PC) are within a defined historical acceptance range. The suggested PC chemical is Brij 35 (CAS No. 9002-92-0) at 100 mg/mL. This concentration should give approximately 30% fluorescein leakage (acceptable range 20-40% fluorescein leakage, i.e. damage to cell layer). The suggested NC chemical is calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS (untreated, blank control).A maximum leakage control should also be included in each run to allow for the calculation of FL20 values. Maximum leakage is determined using a control insert without cells.Determination of fluorescein permeability30. Immediately after removal of the test and control substances, 400μL of 0.1 mg/mL sodium-fluorescein solution (0.01% (w/v) in calcium-containing [at a concentration of 1.0-1.8 mM], phenol red-free, HBSS) is added to the Millicell-HA inserts. The cultures are kept for 30 minutes at room temperature. At the end of the incubation with fluorescein, the inserts are carefully removed from each well. Visual check is performed on each filter and any damage which may have occurred during handling is recorded.31. The amount of fluorescein that leaked through the monolayer and the insert is quantified in the solution which remained in the wells after removal of the inserts. Measurements are done in a spectrofluorometer at excitation and emission wavelengths of 485 nm and 530 nm, respectively. The sensitivity of the spectrofluorometer should be set so that there is the highest numerical difference between the maximum FL (insert with no cells) and the minimum FL (insert with confluent monolayer treated with NC). Because of the differences in the used spectrofluorometer, it is suggested that a sensitivity is used which will give fluorescence intensity > 4000 at the maximum fluorescein leakage control. The maximum FL value should not be6© OECD, (2012)OECD/OCDE 460 greater than 9999. The maximum fluorescence leakage intensity should fall within the linear range of the spectrofluorometer used.Interpretation of results and Prediction model32. The amount of FL is proportional to the chemical-induced damage to the tight junctions. The percentage of FL for each tested concentration of chemical is calculated from the FL values obtained for the test substance with reference to FL values from the NC (reading from the confluent monolayer of cells treated with the NC) and a maximum leakage control (reading for the amount of FL through an insert without cells).The mean maximum leakage fluorescence intensity = xThe mean 0% leakage fluorescence intensity (NC) = yThe mean 100% leakage is obtained by subtracting the mean 0% leakage from the mean maximum leakage,i.e. x - y = z33. The percentage leakage for each fixed dose is obtained by subtracting the 0% leakage to the mean fluorescence intensity of the three replicate readings (m), and dividing this value by the 100% leakage, i.e. %FL = [(m-y) / z] x 100%, where:m = the mean fluorescence intensity of the three replicate measurements for the concentration involved% FL = the percent of the fluorescein which leaks through the cell layer34. The following equation for the calculation of the chemical concentration causing 20% FL should be applied:FL D = [(A-B) / (C-B)] x (M C –M B) + M BWhere:D = % of inhibitionA = % damage (20% fluorescein leakage)B = % fluorescein leakage < AC = % fluorescein leakage > AM C = Concentration (mg/mL) of CM B = Concentration (mg/mL) of B35. The cut-off value of FL20 for predicting chemicals as ocular corrosives/severe irritants is given below:7© OECD, (2012)460OECD/OCDE36. The FL test method is recommended only for the identification of water soluble ocular corrosives and severe irritants (UN GHS Category 1, EU CLP Category 1, U.S. EPA Category I) (see paragraphs 1 and 10).37. In order to identify water soluble chemicals (substances and mixtures) (4) (7) (8) as "inducing serious eye damage" (UN GHS/EU CLP Category 1) or as an "ocular corrosive or severe irritant" (U.S. EPA Category I), the test substance should induce an FL20 value of ≤ 100 mg/mL.Acceptance of results38. The mean maximum fluorescein leakage value (x) should be higher than 4000 (see paragraph 31), the mean 0% leakage (y) should be equal or lower than 300, and the mean 100% leakage (z) should fall between 3700 and 6000.39. A test is considered acceptable if the positive control produced 20% to 40% damage to the cell layer (measure as % fluorescein leakage).DATA AND REPORTINGData40. For each run, data from individual replicate wells (e.g. fluorescence intensity values and calculated percentage FL data for each test substance, including classification) should be reported in tabular form. In addition, means ± SD of individual replicate measurements in each run should be reported.Test Report41. The test report should include the following information:Test and Control Substances-Chemical name(s) such as the structural name used by the Chemical Abstracts Service (CAS), followed by other names, if known;-Chemical CAS number, if known;-Purity and composition of the substance or mixture (in percentage(s) by weight), to the extent this information is available;-Physical-chemical properties relevant to the conduct of the study (e.g. physical state, volatility, pH, stability, water solubility, chemical class);-Treatment of the test/control substance prior to testing, if applicable (e.g. warming, grinding);-Storage conditions;Justification of the Test Method and Protocol Used-Should include considerations regarding applicability domain and limitations of the test method;Test Conditions8© OECD, (2012)OECD/OCDE 460 -Description of cell system used, including certificate of authenticity and the mycoplasma status of the cell line;-Details of test procedure used;-Test substance concentration(s) used;-Duration of exposure to the test substance;-Duration of incubation with fluorescein;-Description of any modifications of the test procedure;-Description of evaluation criteria used;-Reference to historical data of the model (e.g. negative and positive controls, benchmark chemicals, if applicable);-Information on the technical proficiency demonstrated by the laboratory;Results-Tabulation of data from individual test substances and controls for each run and each replicate measurement (including individual results, means and SDs);-The derived classification(s) with reference to the prediction model and/or decision criteria used;-Description of other effects observed;Discussion of the Results-Should include considerations regarding a non-conclusive outcome (paragraph 35: FL20 > 100 mg/mL) and further testing;Conclusions9© OECD, (2012)460OECD/OCDELITERATURE1.UN (2009), United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Third revised edition, New York & Geneva: United Nations Publications.ISBN: 978-92-1-117006-1. Available at:[/trans/danger/publi/ghs/ghs_rev03/03files_e.html]2.EC (2008), Regulation (EC) No 1272/2008 of the European Parliament and of the Council of16 December 2008 on classification, labelling and packaging of substances and mixtures,amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal of the European Union L353, 1-1355.3.U.S. EPA (1996), Label Review Manual: 2nd Edition, EPA737-B-96-001, Washington DC:U.S. Environmental Protection Agency.4.EC-ECVAM (2009), Statement on the scientific validity of cytotoxicity/cell-function based invitro assays for eye irritation testing. Available under Publications at: [http://ecvam.jrc.it/index.htm]5.Scott, L. et al. (2010), A proposed eye irritation testing strategy to reduce and replace in vivostudies using Bottom-Up and Top-Down approaches, Toxicol. In Vitro 24, 1-9.6.OECD (2002), Test No. 405: Acute Eye Irritation/Corrosion, OECD Guidelines for theTesting of Chemicals, Section 4, OECD Publishing. doi: 10.1787/9789264070646-en7.EC-ECVAM (1999), INVITOX Protocol 71: Fluorescein Leakage Test, Ispra, Italy:European Centre for the Validation of Alternative Methods (ECVAM). Available at: [http://ecvam-dbalm.jrc.ec.europa.eu]8.EC-ECVAM (2008), Fluorescein Leakage Assay Background Review Document as anAlternative Method for Eye Irritation Testing. Available under Validation Study Documents, Section Eye Irritation at: [http://ecvam.jrc.it/index.htm]9.OECD (2005), Guidance Document on the Validation and International Acceptance of Newor Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No. 34. OECD, Paris. Available at: [/env/testguidelines]10© OECD, (2012)。
植物原生质体在分子细胞生物学研究中的应用
植物原生质体在分子细胞生物学研究中的应用作者：肖政徐艳琴罗念周银来源：《广西植物》2020年第04期摘要：植物原生质体是去除了细胞壁的裸露细胞，其具有细胞全能性，现广泛应用于植物分子细胞生物学的研究中，可以大大缩减实验周期，并有助于得到体内实验的實时检测数据。
该文除了介绍植物原生质体的提取和纯化方法外，还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。
植物原生质体还可用于基因表达模式的实时检测，并作为生物反应器的受体细胞进行代谢物的体外生产。
此外，还对当前该技术所面临的瓶颈进行了分析，为植物原生质体在分子细胞生物学领域的应用提供帮助，为技术的优化和推广提供参考。
关键词：植物原生质体，瞬时转化，亚细胞定位，细胞融合，实时检测中图分类号： Q942 文献标识码： A文章编号： 1000-3142（2020）04-0576-07Abstract： Plant protoplasts are naked cells without cell walls. They have been extensively applied in the researches of plant molecular and cell biology for their totipotency， which could greatly shorten the experimental periods and help to get massive effective and real-time experimental detection data in vivo. In this article， in addition to introduce the purification of plant protoplasts，we mainly summarized the application of plant protoplasts in the respects of transient transformation， subcellular localization， cell fusion and macromolecular complex interaction. Plant protoplasts could also be used to survey the expression pattern of gene in real-time detection， as well as the target cells for the production of metabolites in bioreactors. Furthermore， we have compared the advantages and disadvantages of plant protoplasts in the current research， which provides new insights into the researches on plant molecular and cell biology. We have also analyzed the difficulties in the application of plant protoplasts， which provides the reference for the optimization and promotion of this technology.Key words： plant protoplasts， transient transformation， subcellular localization， cell fusion， real-time detection植物原生质体是指通过酶解或者机械的方式去除植物细胞壁所获得的细胞。
N-乙酰半胱氨酸对软骨细胞基质降解和骨关节炎的作用研究
N-乙酰半胱氨酸对软骨细胞基质降解和骨关节炎的作用研究张卫华1,张建业1,叶恒1,李永寿2 *(1.武汉市汉阳医院骨科，湖北 武汉 430050；.湖北医药学院基础医学院，湖北 十堰 442000)摘要：目的探讨N-乙酰半胱氨酸(N-acetylcysteine,NAC )对软骨细胞基质降解和骨关节炎的作用。
方法体 外建立白细胞介素(interleukin, IL)-1|3诱导的软骨细胞模型，采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium ,MTT )法和碘化毗啶染色检测细胞活性;采用商用细胞活性氧(reactive oxygen species , ROS)试剂盒、超氧化物检测(superoxide dismutase ,SOD )试剂盒、总谷胱甘肽过氧化物酶(glutathione peroxidase , GPX )检测试剂盒检测细胞内ROS 、SOD 及GPX 含量；采用蛋白印迹法检测细胞基质金属蛋白酶(matrix metalloprotinase , MMP )3、MMP-9、MMP-13及ADAMTS 4的蛋白表达水平;采用阿尔新蓝染色法检测软骨细胞基质;体内采用右膝关节前交叉韧带切断术建立骨关节炎大鼠模型，检测大鼠的机械性疼痛阈值和膝关节肿胀直径;采用反转录聚合酶链式反应(reversetranscription-polymerase chain reaction,RT-PCR)法检测大鼠滑膜组织 MMP-4、MMP-9 和 MMP-13 的表达水平；采用番红固绿染色法检测大鼠滑膜组织的病理学变化。
结果在体外，NAC 能够显著抑制IL1|3诱导的软骨细胞ROS 、SOD 和GPX 产生，同时下调MMP-4.MMP-9.MMP-13和ADAMTS 4的蛋白表达，除此之外,NAC 也能够抑制软骨细胞基质降解。
在体内,NAC 显著增加骨关节炎(osteoarthrosis, OA )大鼠的机械性痛阈值，减小OA 大鼠的关节肿胀；RT-PCR 结果表明NAC 显著下调OA 大鼠滑膜组织MMP-4.MMP-9和MMP-13的表达水平;番红固绿染色结果表明NAC 显著缓解OA 大鼠的关节基质破坏和降解。
主要荧光素一览表之欧阳语创编
（1）荧光素类Fluorescein标准荧光素（Reference standard）之一,在其基础上进行结构改造,可产生一系列荧光素衍生物。
Fluorescein适用于Argon-ion Laser的488nm光谱线，有相对高的荧光吸收，较好的荧光产率以及良好的水溶性。
标记蛋白时通常不会产生蛋白沉淀。
与其他荧光素类衍生物一样，Fluorescein具有光淬灭率高，pH敏感性强与发射波谱宽的缺点。
主要应用于聚焦激光扫描微阵列（Confocal laser scanning microscopy）和流式细胞计应用（Flow cytometry）。
FITC异硫氰酸荧光素，Fluorescein isothiocyanate，是荧光素衍生物的一种，5-FITC较6-FITC更经常使用。
FITC的异硫氰酸基能与氨基反应，可用于标记氨基修饰DNA，一旦形成，产物极为稳定。
适用于Argon-ion Laser的488nm光谱线，Abs/Em=492/519nm（pH=9.0）。
与蛋白的结合力也强。
FITC具有荧光素衍生物的普遍特性。
在水中易变坏，不能长久保存。
FITC-Oligo 广泛用于杂交探针；FITC-多肽用于Edman降解蛋白测序；FITC也经常被用于蛋白电泳检测（即使是毛细管电泳）和荧光能量激发转移测试。
FAM羧基荧光素，Carboxyfluorescein，是荧光素衍生物的一种，5-FAM较6-FAM更经常使用。
Carboxyfluorescein-5-succimidyl ester，即5-FAM （NHS）广泛存在于荧光标记试剂盒。
与FITC相比，FAM与氨基反应更快，产物也更稳定，但FITC结合蛋白的量更大且进程更易于控制。
FAM也适用于Argon-ion Laser的488nm光谱线，Abs/Em=492/518nm（pH=9.0），具有荧光素衍生物的普遍特性，在水中稳定。
5-FAM主要应用于DNA自动测序中，标记其中的d/ddCTP（PE公司），也经常用于PCR产物定量，核酸探针等。
Fluorescein Isothiocyanate产品说明书
FLUORESCEIN ISOTHIOCYANATEProduct Numbers: F4274, Isomer I F7250, Isomer IF1628, Isomer I on Celite F4002, Isomer IIF3651, Mixed IsomersStorage Temperature 2-8 °CSynonym: FITCCAS #: 3326-32-7Product DescriptionAppearance: powderMolecular Formula: C 21H 11NO 5S Molecular Weight: 389.4 Excitation: λmax = 495 nm Emission: λmax = 525 nmThe full absorbance spectrum has been reported.1Fluorescein isothiocyanate (FITC) is widely used to attach a fluorescent label to proteins vi a the amine group. The isothiocyanate group reacts with amino terminal and primary amines in proteins. It has been used for the labeling of proteins including antibodiesand lectins.2-6Isomer I has the thiocyanate group on the 4 carbon of the benzene ring, whereas isomer II has thethiocyanate on the 5 carbon. The two isomers areindistinguishable spectrally, either by wavelength or intensity. Isomer I is more easily isolated in pure form, so is less expensive. This may explain why isomer I is more commonly used for labeling. For many purposes, however, the mixed isomers of FITC will be perfectly suitable.7 The geometry of binding to biomolecules and resulting properties related to elution under electrophoretic or HPLC conditions may, however, require the use of a single pure isomer. The coupling procedure is straightforward experimentally, and the reaction is rapid. A method for determining the degree of substitution is reported.2 A similar method is described in the Sigma bulletin for the FluoroTag™FITC conjugation kit, FITC-1.Adsorbing FITC onto Celite (diatomaceous earth), offered as product F1628, has been reported to increase the efficiency of dispersing FITC in a protein solution.8 FITC on Celite (isomer I) reportedly reacts very quickly with proteins,8 so much faster that antibody titer may be lost by overacylation of the free amino groups. Sigma has not confirmed this claim; however, the FITC on Celite does permit weighing manageable quantities when working with small amounts of protein and avoids the use of organic solvents. FITC on Celite has been used for the labeling of fibrinogen.9 Preparation InstructionsFITC is tested for solubility and solution appearance at 1 mg/ml in acetone. It is soluble in anhydrous dimethyl sulfoxide (DMSO) at 5 mg/ml.10 It is soluble in water at less than 0.1 mg/ml in water, at 20 mg/ml in ethanol and at 9 mg/ml in 2-methoxyethanol.1 An organic solvent for stock solution is advised, since FITC decomposes in water. FITC is diluted in basic buffer for coupling procedures immediately prior to use.11 Storage/StabilityThe products are light-sensitive, and should be stored dry and in the dark at 2 °C to 8 °C.ProcedureLabeling of Protein with FITC3, 5, 61. Prepare a solution of at least 2 mg/ml of protein in0.1 M sodium carbonate buffer, pH 9.Notes: Do not store sodium carbonate-bicarbonate buffer more than 1 week at 0-5 °C. The pH of thebuffer may change upon storage. It is advised that fresh buffer be made just before use.The protein to be conjugated should be free ofcontaminating proteins, and protein solutionsshould not be prepared in buffers containingsodium azide or amines such as Tris or glycinesince they inhibit the labeling reaction. If the buffer contains amines or sodium azide, dialyze proteinsolution against PBS, pH 7.4, overnight at 0 - 5 °C.Avoid dialysis at high pH values (> 8.0-8.5) as this may be harmful to some proteins.2. Dissolve the FITC in anhydrous DMSO at 1 mg/ml.Note: This should be prepared fresh for eachlabeling reaction.3. For each 1 ml of protein solution, add 50 µl of FITCsolution, very slowly in 5 µl aliquots while gentlyand continuously stirring the protein solution.4. After all the required amount of FITC solution hasbeen added, incubate the reaction in the dark for 8 hours at 4 °C.5. Add NH4Cl to a final concentration of 50 mM andincubate for 2 hours at 4 °C.6. Add xylene cyanol to 0.1% and glycerol to 5%.7. Separate the unbound FITC from the conjugate bygel filtration using a fine-sized gel matrix with anexclusion limit of 20,000 to 50,000 (for globularproteins such as antibodies). With the column flow stopped, carefully layer the reaction mixture ontothe top of the column. Then open the column,allowing the reaction mixture to flow into thecolumn. Just as it all enters the column bed,carefully add PBS to the top of the column andconnect to a buffer supply.Two bands will form on the column. The fastermoving band, which is the conjugated protein,elutes first and can usually be seen under roomlight. The slower moving band is the unreacted(free) FITC and xylene cyanol and will elute onlywith subsequent PBS washes.8. Store the conjugate at 4 °C in the column buffer ina light-proof container. Sodium azide may be addedas a preservative (final concentration 15 mM). If the protein concentration is low (< 1 mg/ml), bovineserum albumin (BSA) may be added to a finalconcentration of 1%.9. The ratio of fluorescein to protein of the product canbe estimated by measuring the absorbance at495 nm and 280 nm. The F/P ratio should bebetween 0.3 and 1.0. Lower ratios will yield lowsignals; higher ratios will give high background.Determination of Fluorescein/Protein Molar Ration (F/P)The F/P molar ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate. To determine this ratio, it is necessary to first determine the absorbance of the conjugate sample at 280 nm and then at 495 nm.Place the conjugate sample in a quartz cuvette. Read the absorbance of the conjugate sample at 280 nm and 495 nm. The absorbance reading of the conjugate sample should be between 0.2 and 1.4 at 280 nm. If the absorbance reading is outside this range, adjust the sample dilution accordingly.For FITC-IgG conjugates only:From the absorbance readings (A280 and A495) of the conjugate sample, calculate the F/P ratio of the conjugate according to the equations:2.77 x A495Molar F/P =A280-(0.35 x A495)The protein concentration of the fluorescein-IgG conjugate is calculated from the following formula: [A280 -(0.35 x A495)]IgG (mg/ml) =1.4Where 1.4 is the A280 of IgG from most species at a concentration of 1.0 mg/ml at pH 7.0.For other FITC-protein conjugates:When any protein other than IgG is conjugated to FITC, use the general formula below, substituting the appropriate values for the particular protein:MW A495/195Molar F/P = x =389 [A280-(0.35 x A495)]/E0.1%A495 x C A280-[(0.35 x A495)]MW x E0.1%280Where: C =389 x 195C is a constant value given for a protein.MW is the molecular weight of the protein.389 is the molecular weight of FITC.195 is the absorption E0.1% of bound FITC at490 nm at pH 13.0.(0.35 X A495) is the correction factor due to the absorbance of FITC at 280 nm.8E0.1% is the absorption at 280 nm of a protein at1.0 mg/ml.F1628 is approximately 10 % FITC (by weight) on CeliteCelite is a trademark of Manville Service Corp. References1. Green, F.J., Sigma-Aldrich Handbook of Stains,Dyes and Indicators, p. 377 (1990).2. Schreiber, A.B., and Haimovich, J., MethodsEnzymol., 93, 147-155 (1983).3. Harlow, E. and Lane, D., Antibodies a LaboratoryManual, pp. 353-355 (Cold Spring HarborLaboratory, 1988).4. Bridges, C.D. and Fong, S.L., Methods Enzymol.,81, 65-77 (1982).5. The, T.H., and Feltkamp, T.E.W., Immunology, 18,865-873 and 875-881 (1970).6. Goding, J.W., J. Immunol. Methods, 13, 215-226(1976).7. NCCLS Approved Standard:ASM-1, 2nd Ed. (June1975). Standard test for labeling efficiency of FITC with BSA (albumin).8. Rinderknecht, H., Experientia, 16, 430 (1960).9. Xia, Z., et al., Br. J. Haematol., 93, 204-214 (1996).10. Current Protocols in Immunol., Coligan, J.E. et al.(Eds.), pp. 5.3.3 (Wiley & Sons).11. McKinney, R.M., et al., Anal. Biochem., 14, 421-428 (1966).AJH 8/08Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。
荧光检测器
Fluorescence detector
Sensitivity of the detection depends on the quantum efficiency of the molecule
分子的量子效率Q：产生荧光的分子在激发态分子中 的比例 .
Large group of biological molecules such as protein with aromatic amino acid residues and signal transduction chemicals such as catecholamines (儿茶酚胺) can have native fluorescence. But the fluorescence efficiency are low.
利用待测荧光和背景光在寿命上的差异消除背景杂散 光的影响，利用短脉冲激光和时间门迭通技术，进行
时间分辨测量，当短寿命杂散光衰减到很弱时，打开
时间门采集荧光。
Mass Spectrometry Detection ( 质谱检测器)
Advantage
• Good sensitivity（LOD: 10-12 mol）
Ultraviolet/ Visible absorbance Detection
Focusing with sapphire ball
在柱上检测中，用蓝宝石球镜聚焦后，可使光束聚焦到0.2nl 体积，提高 入射光强。
毛细管圆柱面类似短焦柱面透镜，使 入射圆形光束变为椭圆形光束，收集困 难，检测线高于HPLC. 几种增加检测灵敏度的方法：
( 激光诱导荧光检测 )
Because of the high intensity，narrow band with and