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View additional product information for Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit, 100 rxn(s) - FAQs (C10635)
34 product FAQs found
•当铜离子在合适的化合价时，点击反应才有效。除了DIBO炔烃-叠氮化物反应外，在缺乏铜离子的条件下，叠氮化物和炔烃不会相互反应。确保在制备之后、二价铜(II)浓度最高时，立即使用点击反应混合物。
•不要使用已经变黄的添加剂缓冲液；其必须无色状态下才有活性。
•为确保TdT酶和点击反应试剂能够到达核内，细胞需要充分地固定和通透。为确保有足量的TdT能进入，组织样品需要用蛋白酶K或其他蛋白水解酶消化。
•部分试剂能结合到铜离子上，并减少其足以催化点击反应的有效浓度。click反应前，不要在任何缓冲液或试剂中引入任何金属螯合剂（例如EDTA、EGTA、柠檬酸盐等）。避免缓冲液和试剂中引入其他可能被氧化或还原的金属离子。进行click反应前，可能需要向细胞或组织样品加入额外的洗涤步骤。
•您可以用新鲜的试剂再次进行点击反应，从而提高信号。将点击反应的时间延长至超过30分钟，并不会提高信号。用新鲜的点击反应试剂再次进行30分钟的孵育，可更有效地提高标记效率。
•自己的细胞可能没有凋亡。准备一份DNase I处理的阳性对照，验证TdT酶反应和点击标记反应是否正确进行。
click反应在叠氮化物和炔烃类间极具选择性。生物体系不可能有其他副反应。任何非特异性背景都是由于染料和多种细胞内组分的非共价连接。Select FX信号增强剂在click反应后减少染料的电荷为基础的非特异性连接方面效果不明显；我们不推荐将其与Click-iT检测试剂一同使用。最佳的减少背景的方法是增加BSA清洗的次数。你应该在同样的过程和检测条件下做无染料或无click反应对照，以证实背景确实是由于染料而不是自发荧光。你同时应该在无TdT酶的对照样品进行完整的click反应来证实click反应信号的特异性。
click反应中的铜离子使得DNA少量变性（虽然没有BrdU检测所需要的程度），这会影响到包括DAPI和Hoechst染色剂在内的DNA染料的结合亲和力。这种影响只会在传统的EdU试剂盒中出现，而Click-iT Plus EdU试剂盒由于采用的铜离子浓度低，不会出现这种现象。
•当铜离子在合适的化合价时，点击反应才有效。除了DIBO炔烃-叠氮化物反应外，在缺乏铜离子的条件下，叠氮化物和炔烃不会相互反应。确保在制备之后、二价铜浓度最高时，立即使用点击反应混合物。
•不要使用已经变黄的添加剂缓冲液；其必须无色状态下才有活性。
•细胞需要充分固定和通透，确保click试剂能充分接触到细胞内已掺入click底物的成分。
•部分试剂能结合到铜离子上，并减少其足以催化点击反应的有效浓度。click反应前，不要在任何缓冲液或试剂中引入任何金属螯合剂（例如EDTA、EGTA、柠檬酸盐等）。避免缓冲液和试剂中引入其他可能被氧化或还原的金属离子。进行click反应前，可能需要向细胞或组织样品加入额外的洗涤步骤。
•你可以用新鲜的实验试剂重复click反应尝试提高信号。增加click反应的时间长于30分钟不会提高低的信号。用新鲜的click反应试剂进行第二次30分钟培养在提高标记上更加有效。
•低信号同时也可能是因为EdU、EU和其他click底物的掺入水平较低。如果无法进行充分交联或使用的固定剂有误，其他掺入到细胞组件的click底物（如AHA、HPG、棕榈酸、叠氮化物等）可能会丢失。对于掺入到细胞膜或脂质的click底物，应避免使用乙醇或丙酮固定剂和透化试剂。
•在click反应时，掺入的click底物必须可被接触；相比于变性蛋白，天然蛋白中掺入的氨基酸模拟物的标记水平可能更低。
•你可能需要优化代谢标记条件，包括模拟物孵育时间和浓度。健康的、传代数不高、不太密集的细胞可能更容易掺入模拟物。如果孵育时间达到关注的细胞数目翻倍的时间，则应在多个整倍时间点设置包含额外剂量的click底物的阳性对照。
click反应在叠氮化物和炔烃类间是非常有选择性的。生物体系不可能有其他副反应。任何非特异性本底都是由于染料和多种细胞组件的非共价结合造成的。在click反应后，Select FX信号增强剂在减少染料非特异性电荷连接方面的作用失效；我们不推荐将其与Click-iT检测试剂一同使用。减少本底干扰的最佳方法是增加BSA洗涤的次数。您应始终在同等的处理和检测条件下做无染料或无click反应对照，以证实本底确系染料而非自发荧光所致。此外，您还可在无EdU 或无-EU，仅含溶剂的对照样本上进行完整的click反应，验证click反应信号的特异性。
不可以，EdU代谢标记试剂必须用于活细胞，但是实际的检测反应必须在固定和通透的样品上进行，因为叠氮化物检测试剂和缓冲液组分是细胞非透过型。
也许可以，但是如果您没有在第一次Click反应中将所有的代谢嵌入EdU完全标记，之后其可能在第二次的TUNEL标记Click反应中被标记，导致细胞凋亡假阳性。将Click-iT EdU 标记与BrdU TUNEL 标记结合会更简单，因为BrdU检测不会与EdU标记的细胞交叉反应。如果您真的希望为增殖和细胞凋亡检测进行双EdU标记，您必须使用新鲜的连接试剂来重复连接反应检测代谢嵌入的EdU，确保进行EdU TUNEL实验前所有的嵌入EdU已经被标记。之后您应该进行一个无–TdT酶EdU TUNEL对照实验来验证TUNEL连接反应没有信号产生。
可以，EdU和BrdU标记能结合起来在培养细胞和体内用于细胞增殖的双重标记。BrdU将会优先的嵌入到DNA，因此先进行EdU培养再进行BrdU培养。当添加BrdU作为第二标记时，不需要从细胞培养的培养基中移除EdU。进行乙醇固定，随后进行BrdU检测方案中需要的DNA变性的一些方法。之后为EdU检测进行连接标记反应，随后进行用于BrdU检测的抗体标记。确保选择一个不和EdU交叉反应的BrdU抗体，例如我们的MoBU-1克隆（货号B35141）。许多BrdU抗体表现出对EdU一定程度的交叉性反应。此处（https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/dual-pulse-labeling-of-cell-proliferation-using-edu-and-brdu-incorporation.html）是使用EdU和BrdU进行双-脉冲标记案例的链接。
我们还没有验证在3D培养体系中使用EdU研究增殖，但是这种试剂适用于体内细胞标记，其同时可预料能够标记3D培养体系中的细胞。文献中有大量关于在3D培养体系中使用这种产品报道；此处有一些引证：
Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation.Proc Natl Acad Sci U S A 110:E5039–E5048.
Derda R, Laromaine A, Mammoto A et al.(2009) Paper-supported 3D cell culture for tissue-based bioassays.Proc Natl Acad Sci U S A 106:18457–18462.
Robertson FM, Ogasawara MA, Ye Z et al.(2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype.J Biomol Screen 15:820–829.
有的，在透化作用步骤前，您可以将甲醛固定和清洗后的样品储存起来。就将细胞保存在PBS中，将容器良好的封闭并密封，4°C储存。细胞应该至少1星期完好。您也可以在连接反应和清洗步骤后将样品储存起来，在第二天进行免疫染色和核染色。
不可以，Click-iT Plus和初代Click-iT试剂盒中的用于连接反应的试剂和检测试剂不可以混用。Click-iT Plus实验使用修饰的甲基吡啶叠氮化物染料且降低铜离子浓度并加入了特别的铜离子保护剂，其将铜离子置于嵌入的炔烃基团，然而初代Click-iT试剂盒使用非修饰叠氮化物染料和高铜离子浓度来进行连接反应。
Click-iT Plus实验使用了一个修饰的甲基吡啶叠氮化物染料且降低铜离子浓度并加入了特别的铜离子保护剂，其将铜离子置于嵌入的炔烃基团位置因而降低了铜对生物分子的损伤。初代Click-iT试剂盒使用非修饰叠氮化物染料和高铜离子浓度来进行连接反应，这可能抑制酶的活性，包括HRP和会淬灭GFP、RFP、mCherry及其他荧光蛋白的荧光，也包括R-藻红蛋白。如果您不希望修改您的抗体染色方案或拥有荧光蛋白表达细胞，就使用Click-iT Plus试剂盒。
可以通过流式细胞仪完成以下细胞健康和活力检测：
细胞凋亡检测：
细胞膜不对称性：膜联蛋白V是结构相关蛋白家族一员，其可以在Ca2+存在的情况下结合磷脂。膜联蛋白V可结合多种磷脂，但是对磷脂酰丝氨酸表现出高度的亲和性。磷脂酰丝氨酸主要存在于细胞膜的内部小叶上；然而，在细胞凋亡早期，观测到磷脂酰丝氨酸转移到外部小叶。这个转移使得在含有Ca2+孵育缓冲液存在的情况下，磷脂酰丝氨酸可与膜联蛋白V结合。凋亡中的细胞可以用膜联蛋白V染色，而正常细胞不会被染色。多种偶联不同荧光基团的膜联蛋白V可供选择。
线粒体健康：细胞凋亡早期的典型特征是线粒体紊乱，同时伴随膜和氧化还原电位改变。我们独家提供了大量可通过流式细胞术分析活细胞内的线粒体活性，同时可最大限度避免细胞功能损伤的荧光探针。
线粒体染色的MitoProbe系列染料（MitoProbe DiOC2(3) 检测试剂盒，货号 M34150；MitoProbe JC-1检测试剂盒，货号M34152；MitoProbe DiIC1(5) 检测试剂盒，货号M34151）为检测细胞凋亡过程出现的线粒体膜电位损失提供了快速、简单和可靠的流式细胞术检测手段。
半胱天冬酶活性： CellEvent Caspase-3/7 Green 流式细胞检测试剂盒（货号C10427）支持对凋亡细胞中活化的caspase-3和 caspase-7进行流式细胞术检测。该试剂盒包含新型荧光底物CellEvent Caspase-3/7 Green检测试剂和SYTOX AADvanced死细胞染色剂，可靶向识别活化的caspase-3和caspase-7的序列。
DNA片段化：细胞凋亡后期的特征是核形态改变，包括DNA片段化、染色质凝缩、核膜降解，核起泡以及DNA链断裂。凋亡过程中DNA片段出现DNA链断裂，可以通过TUNEL（末端脱氧核苷酸转移酶dUTP缺口末端标记）检测进行分析。APO-BrdU TUNEL检测（货号A23210）是一种双色检测方法，可通过成像或流式细胞术标记DNA断裂和细胞总DNA，检测细胞凋亡情况。
核染色质凝缩：细胞凋亡后期的特征是核形态改变，包括DNA片段化，染色质凝缩，核膜降解，核起泡以及DNA链断裂。凋亡的细胞表现出核染色质凝结增加。由于核染色质凝结，细胞透过性核酸染色剂发出较高的荧光，从而能够结合传统死细胞染色剂来区分凋亡细胞。
Vybrant细胞凋亡检测试剂盒#5，Hoechst 33342/Propidium Iodide（货号V13244）基于凋亡细胞染色质的压缩状态的荧光检测，为凋亡提供了一个快速和方便的检测手段。染色质凝结&膜渗透死细胞凋亡试剂盒包含Hoechst 33342、YO-PRO-1以及PI染料, 用于流式细胞仪（货号V23201）检测凋亡细胞中核染色质凝结和质膜通透性的变化。
细胞周期分析：
活细胞检测： Vybrant DyeCycle系列染料为活细胞周期分析提供了稳定的低毒性荧光染料，提供405 nm（货号V35003）、488 nm （货号V35004）、532 nm（货号V35005）或 633 nm（货号V10309和V10273）三种激发峰选择。染料毒性低，染色的细胞可储存和培养，或进行功能分析。
固定细胞检测：用FxCycle Violet染色剂（货号F10347）、SYTOX AADvanced 死细胞染色试剂盒（货号S10349）或FxCycle Far Red染色剂（货号F10348）分析细胞周期，为简单固定的细胞周期分析提供了多色选择。
细胞增殖：
染料稀释检测细胞增殖：染料稀释检测细胞增殖依赖细胞膜透过性荧光分子。染料进入到细胞后，共价结合蛋白质的氨基基团，导致染料长期保留在细胞中。通过随后的细胞分裂，每个子代细胞大约会分到亲代一半的荧光。采用流式细胞仪对细胞群的荧光强度进行分析，可以某个细胞或细胞群自标记之后的增殖情况，判定其传代次数。CellTrace荧光染色剂可在不影响细胞形态和生理功能的条件下，在体内或体外追踪传代情况。目前尚未发现该染色剂对细胞增殖活性或细胞生物学功能有影响。染色后，染料可在细胞内稳定保留若干天。可用于流式细胞仪的试剂盒包括CellTrace CFSE细胞增殖试剂盒（货号No. C34554）CellTrace Violet细胞增殖试剂盒（货号 C34557）和CellTrace Far Red细胞增殖试剂盒（货号 C34564）。
DNA合成检测：测定新合成的DNA是准确分析某个细胞或细胞群细胞增殖情况的方法。基于DNA合成的细胞增殖检测可根据掺入的修饰核苷，测定DNA合成速率。Click-iT Plus EdU细胞增殖检测利用了click化学试剂和修饰的核苷EdU，为BrdU染色提供了出色的替代方法，可用于检测和定量新合成的DNA数量。Pacific Blue（货号C10636）、Alexa Fluor 488（货号C10632和C10633）和Alexa Fluor 647（货号C10634和C10635）可用于Click-iT Plus EdU细胞增殖检测。
活力检测：
死细胞很容易与很多试剂非特异性结合，从而给出假阳性结果。因此，从流式细胞仪数据中排除死细胞，是有助于确保结果和分析准确性的关键步骤。
不能固定膜通透染色剂：SYTOX死细胞染色剂（货号S34857、S34860、S34861、S34859和S34862）不能穿过完整的细胞膜，与dsDNA结合后发出更强的荧光，从而成为我们最明亮的几种死细胞染色剂之一。非细胞通透性的经典DNA结合染料包括碘化丙啶（货号P21493）和7-AAD（货号A1310）。这两种染料已经被广泛用于流式细胞仪活性分析。CellTrace钙黄绿素AM染料可被动运输进入到贴壁和非贴壁细胞。这些细胞通透性酯酶底物可作为测定酶活性（激发荧光的必要条件）和细胞膜完整性的（在胞内保留荧光产物的必要条件）的活力探针。目前供应的蓝色（货号 C34853）、紫色（货号C34858）和绿色（货号C34852）荧光染料是活细胞短时染色的理想染料，且可用于多重流式细胞术试验。
可固定细胞活性染色剂：LIVE/DEAD可固定死细胞染色剂是可固定的细胞活性染料，有助于准确评估固定和/或通透后样品中细胞活性。LIVE/DEAD固定死细胞染色试剂盒基于荧光活性染料与细胞蛋白（胺基）的反应。这些染料不能透过活细胞膜，因此仅仅细胞表面蛋白可和染料反应，导致染色暗淡。活性染料可以透过死细胞损毁的细胞膜，将内部和外部的胺基染色，导致更加强烈的染色效果。LIVE/DEAD固定死细胞染色试剂盒可提供八单通道颜色，适用于三种包装规格的UV、405、488、532、561或633 nm激光，可满足您的试验需要。
Alexa Fluor 647 has an excitation/emission maxima of 650/670 nm.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Click reactions have several general characteristics: the reaction is efficient, no extreme temperatures or solvents are required, the reaction is complete within 30 minutes, the components of the reaction are bio-inert, and perhaps most importantly, no side reactions occur-the label and detection tags react selectively and specifically with one another. This final point is a key advantage of this powerful detection technique; it is possible to apply click chemistry-labeled molecules to complex biological samples and detect them with unprecedented sensitivity due to the extremely low background of the reaction.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
One may store the sample after fixation overnight in PBS at 4oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.
For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.
Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The presence of EDTA in the cell buffer/media would not be an issue for EdU incorporation and it should be mostly gone from the sample after fixation and permeabilization. EDTA must not be present during the click reaction.
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The middle peak would most likely be from EdU-incorporated cells that have divided, each daughter cell receiving roughly half the incorporated EdU. To avoid this, we recommend shortening the incubation time of EdU with the cells.
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We do not recommend doing this. The signal provided with the imaging kits may be too high for use on a flow cytometer.
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The click reaction is only effective when copper is in the appropriate valency. Azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the TdT enzyme and click reagents to have access to the nucleus. Tissue samples require digestion with proteinase K or other proteolytic enzymes for sufficient TdT access.
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be o xidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Your cells may not be apoptotic. Prepare a DNase I-treated positive control to verify that the TdT enzymatic reaction and click labeling reaction are working correctly.
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The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You should also perform the complete click reaction on a no-TdT enzyme control sample to verify the specificity of the click reaction signal.
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The copper in the click reaction denatures DNA to a small extent (although not as much as is required for efficient BrdU detection), which can affect the binding affinity of DNA dyes including DAPI and Hoechst stain. This effect should only be apparent with the classic EdU kits and not the Click-iT Plus EdU kits, which use a lower copper concentration.
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The click reaction is only effective when copper is in the appropriate valency. Except for the DIBO alkyne-azide reaction, azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the click reagents to have access to intracellular components that have incorporated the click substrate(s).
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be oxidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Low signal can also be due to low incorporation of EdU, EU, or other click substrates. Other click substrates (e.g., AHA, HPG, palmitic acid, azide, etc.) incorporated into cellular components may have been lost if not adequately cross-linked in place or if the wrong fixative was used. For click substrates that are incorporated into the membrane or lipids, you should avoid the use of alcohol or acetone fixatives and permeabilizing agents.
The incorporated click substrate must be accessible at the time of the click reaction; labeling of incorporated amino acid analogs may be lower in native proteins relative to denatured proteins.
You may need to optimize the metabolic labeling conditions including analog incubation time or concentration. Cells that are healthy, not too high of a passage number and not too crowded may incorporate the analog better. You may create a positive control by including extra doses of the click substrate during multiple time points during an incubation time that spans or closely spans the doubling time of the cell type of interest.
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The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You can also perform the complete click reaction on a carrier solvent-only, no EdU or no-EU control to verify the specificity of the click reaction signal.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
No, the EdU metabolic labeling reagent must be used on live cells, but the actual click detection reaction must be performed on fixed and permeabilized samples, as the azide detection reagents and buffer components are cell impermeant.
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It is possible, but if you have not completely labeled all of the metabolically incorporated EdU in the first click reaction, then it will be labeled in the second click reaction for TUNEL labeling, leading to false positives for apoptotic cells. It would be simpler to combine Click-iT EdU labeling with BrdU TUNEL labeling, as BrdU detection will not cross-react with EdU labeled cells. If you really wish to perform a double EdU labeling for both proliferation and apoptosis detection, then you should repeat the click reaction to detect the metabolically incorporated EdU using fresh click reagents to ensure that all of the incorporated EdU is labeled before performing the EdU TUNEL assay. You should then perform a control no-TdT enzyme EdU TUNEL assay to verify that there is no signal generated with the TUNEL click reaction.
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Yes, EdU and BrdU labeling can be combined for dual-pulse labeling of cell proliferation in cultured cells and in vivo. BrdU will be preferentially incorporated into DNA, so perform the EdU incubation first followed by the BrdU incubation. Removal of EdU from the media is not required in cultured cells when BrdU is added as the second label. Perform an alcohol fixation followed by some method of DNA denaturation as required for the BrdU detection protocol and then perform the click labeling reaction for detection of EdU followed by antibody labeling for detection of BrdU. Be sure to select a BrdU antibody that does not have cross-reactivity to EdU, such as our MoBU-1 clone (Cat. No. B35141). Many BrdU antibodies have been shown to have some amount of cross-reactivity with incorporated EdU. Here is a link (http://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/dual-pulse-labeling-of-cell-proliferation-using-edu-and-brdu-incorporation.html) to an example protocol for dual-pulse labeling using EdU and BrdU.
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We have not validated the use of EdU for proliferation in 3D culture systems, but as this reagent is compatible for labeling cells in vivo, it is also expected to label cells in 3D culture systems. There are a number of reports in the literature that use this product in 3D culture systems; here are some citations:
Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci U S A 110:E5039-E5048.
Derda R, Laromaine A, Mammoto A et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc Natl Acad Sci U S A 106:18457-18462.
Robertson FM, Ogasawara MA, Ye Z et al. (2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype. J Biomol Screen 15:820-829.
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Yes, you can store samples after fixing in formaldehyde and washing, before the permeabilization step. Just keep the cells in PBS, cover and seal the container well, and store at 4 degrees C. The cells should be fine for at least a week. You can also store the samples after the click reaction and wash steps and then perform any immunostaining and nuclear counterstaining on the following day.
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No, the detection reagent and reagents necessary to perform the click reaction cannot be intermixed between the Click-iT Plus and original Click-iT kits. The Click-iT Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the click reaction, while the original Click-iT kits use an unmodified azide dye and higher copper concentrations to perform the click reaction.
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The Click-iT Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the incorporated alkyne group and thus minimizes copper damage to biomolecules. The original Click-iT kits use an unmodified azide dye and higher copper concentrations to perform the click reaction, which may inactivate enzymes, including HRP, and will quench the fluorescence of GFP, RFP, mCherry and other fluorescent proteins, as well as R-phycoerythrin. If you do not wish to modify your antibody staining protocol or have fluorescent protein-expressing cells, then use the Click-iT Plus kits.
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The following cell health and viability assays can be performed by flow cytometry :
-Apoptosis Assays:
Membrane Asymmetry: Annexin V is a member of a family of structurally related proteins that bind phospholipids in the presence of Ca2+. Annexin V binds several phospholipids, but shows highest affinity for phosphatidylserine.
Phosphatidylserine is normally found in the inner leaflet of the cell membrane; however, in the early stages of apoptosis, phosphatidylserine is observed to translocate to the outer leaflet. This translocation makes phosphatidylserine available for annexin V binding in the presence of Ca2+ containing incubation buffer. Cells undergoing apoptosis will stain with annexin V, while normal cells will not. annexin V is available conjugated with a wide range of fluorophores.
Mitochondrial Health: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We exclusively offer a number of fluorescent probes for analyzing mitochondrial activity in live cells by flow cytometry, with minimal disruption of cellular function.
The MitoProbe family of mitochondrial stains (MitoProbe DiOC2(3) Assay Kit, Cat. No. M34150, MitoProbe JC-1 Assay Kit, Cat. No. M34152, and MitoProbe DiIC1(5) Assay Kit, Cat. No. M34151) provides quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis.
Caspase Activity: The CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat. No. C10427) enables flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent Caspase-3/7 Green Detection Reagent which targets the recognition sequence for activated caspase-3 and caspase-7, as well as SYTOX AADvanced Dead Cell Stain.
DNA Fragmentation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. DNA fragmentation that occurs during apoptosis produces DNA strand breaks, and can be analyzed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The APO-BrdU TUNEL assay (Cat. No. A23210) is a two-color assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by imaging or flow cytometry.
Nuclear Chromatin Condensation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. Cells undergoing apoptosis display an increase in nuclear chromatin condensation. As the chromatin condenses, cell-permeable nucleic acid stains becomes hyperfluorescent, thus enabling the identification of apoptotic cells when combined with a traditional dead-cell stain. The Vybrant Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Cat. No. V13244) provides a rapid and convenient assay for apoptosis based on fluorescence detection of the compacted state of the chromatin in apoptotic cells. The Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO-1, and PI dyes, for flow cytometry (Cat. No. V23201) detects apoptotic cells with changes in nuclear chromatin condensation and plasma membrane permeability.
-Cell Cycle Analysis:
Live cell assays: The Vybrant DyeCycle family of dyes offers robust fluorescent dyes for live-cell cycle analysis with limited cytotoxicity using 405 nm (Cat. No. V35003), 488 nm (Cat. No. V35004), 532 nm (Cat. No. V35005), or 633 nm (Cat. Nos. V10309 and V10273) excitation. The dyes have low cytotoxicity, allowing stained cells to be sorted and otherwise cultured or assessed with functional assays after staining.
Fixed cell assays: Analyzing cell cycle using FxCycle Violet Stain (Cat. No. F10347), SYTOX AADvanced Dead Cell Stain Kit (Cat. No. S10349) or FxCycle Far Red Stain (Cat. No. F10348) allows for multiple color options for simplified fixed cell cycle analysis.
-Cell Proliferation:
Dye dilution assays for cell proliferation: Dye dilution assays for cell proliferation rely on cell membrane–permeant fluorescent molecules. Upon entry into the cell, the dye will covalently bind to amine groups on proteins, resulting in long-term dye retention within the cell. Through subsequent cell divisions, each daughter cell receives approximately half the fluorescence of the parent. Analysis of the fluorescence intensities of cell populations by flow cytometry enables determination of the number of generations through which a cell or population has progressed since the label was applied. CellTrace fluorescent stains can be used without affecting morphology or physiology to trace generations in vivo or in vitro. There is no known effect on proliferative ability or biology of cells and they are well retained in cells for several days post-stain. Available kits for flow cytometry include CellTrace CFSE Cell Proliferation Kit (Cat. No. C34554), CellTrace Violet Cell Proliferation Kit (Cat. No. C34557), and CellTrace Far Red Cell Proliferation Kit (Cat. No. C34564).
DNA Synthesis Assays: Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. DNA synthesis–based cell proliferation assays measure the rate of new DNA synthesis based on incorporation of modified nucleosides. The Click-iT Plus EdU cell proliferation assay utilizes the power of click chemistry and the modified nucleoside EdU to provide a superior alternative to BrdU staining for detecting and quantitating newly synthesized DNA. The Click-iT Plus EdU cell proliferation assay is available with Pacific Blue (Cat. No. C10636), Alexa Fluor 488 (Cat. Nos. C10632 and C10633), and Alexa Fluor 647 (Cat. Nos. C10634 and C10635).
-Viability Assays:
Dead cells often give false positive results, as they tend to bind non-specifically to many reagents. Therefore, removing dead cells from your flow cytometry data is a critical step to help ensure accurate results and analysis.
Non-fixable Membrane Permeability Stains: SYTOX Dead Cell Stains (Cat. Nos. S34857, S34860, S34861, S34859, and S34862) do not cross intact cell membranes, and they exhibit increased fluorescence upon dsDNA binding, making them some of our most brilliant dead cell stains. Cell-impermeant classic DNA-binding dyes include propidium iodide (Cat. No. P21493) and 7-AAD (Cat. No. A1310). Both of these dyes have been used extensively for viability assays in flow cytometry. CellTrace Calcein AM dyes can be passively loaded into adherent and nonadherent cells. These cell-permeant esterase substrates serve as viability probes that measure both enzymatic activity, which is required to activate their fluorescence, and cell membrane integrity, which is required for intracellular retention of their fluorescent products. Available with blue (Cat. No. C34853), violet (Cat. No. C34858), and green (Cat. No. C34852) fluorescence, these dyes are ideal for short-term staining of live cells and can be used in multiplexed flow cytometry experiments.
Fixable Viability Stains: The LIVE/DEAD Fixable Dead Cell Stains are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization. LIVE/DEAD Fixable Dead Cell Stain Kits are based on the reaction of a fluorescent reactive dye with cellular proteins (amines). These dyes cannot penetrate live-cell membranes, so only cell-surface proteins are available to react with the dye, resulting in dim staining. The reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, resulting in more intense staining. LIVE/DEAD Fixable Dead Cell Stain Kits are available in eight single-channel colors available for UV, 405, 488, 532, 561, or 633 nm lasers in three packaging sizes to match your experiment.
Other than the EdU (Component A), DMSO (Component C), and Click-iT EdU buffer additive (Component F or G), all other components in the respective kits are not interchangeable. The types of reagents and amounts provided per kit were optimized either for imaging or flow cytometry. Using the imaging reagents may result in an excessive level of signal for flow cytometry detection and using the flow cytometry reagents may result in sub-optimal signal for imaging.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.