Search
Search
View additional product information for Click-iT™ Plus EdU Alexa Fluor™ 350 Flow Cytometry Assay Kit - FAQs (C10645)
25 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。
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 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.
Find additional tips, troubleshooting help, and resources within our Labeling Chemistry Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.