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View additional product information for Click-iT™ TUNEL Alexa Fluor Imaging Assays for Microscopy & HCS - FAQs (C10245, C10247, C10246)
24 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。
可以，额外的末端脱氧核苷酸转移酶（rTdT）可以购买。货号为10533-065或10533-075。额外的TdT反应缓冲液可以购买，货号为16314-015。
我们没有在3D培养体系中使用过EdU TUNEL检测调亡，但是因为这种试剂适用于体内细胞标记，所以它也有希望可以标记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.
大部分组织样品需要充分消化15分钟。最佳的培养时间根据组织类型和厚度而不同。我们已经观测到脑组织比其他组织需要更长的蛋白酶处理时间。
我们在5–20 µM 厚度的老鼠肠、肾脏、肝脏、心脏和结肠FFPE切片中验证了Click-iT Plus TUNEL for In Situ Apoptosis Detection实验
初代Click-iT TUNEL实验试剂盒针对细胞培养样品优化，也可用于组织样品。我们建议对组织样品使用Click-iT Plus TUNEL for In Situ Apoptosis Detection实验试剂盒；用于组织的Click-iT Plus TUNEL for In Situ Apoptosis Detection实验试剂盒的实验方案针对组织样本优化。通过改进方案可以提高TdT酶进入多层细胞组织样品的效果。另外，相比于Click-iT Plus TUNEL试剂盒，我们发现初代Click-iT TUNEL试剂盒可能会在组织样品出现高度非特异性结合和点状染色。有一个增加组织透过性较好的方法是用蛋白酶K消化然后甲醛中再固定样品以取代去污剂透化作用步骤。Click-iT Plus TUNEL试剂盒手册第三部分的组织固定和透化作用方案可用于初代Click-iT TUNEL实验处理组织样品。胃蛋白酶和其他的蛋白水解酶同时也可用于提高组织透过性。
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。
•可测定来自单个细胞的数据。
•可从大量细胞中获得数据，产生细胞群的丰富统计学分析结果。
•由于可测定单个细胞，能够揭示种群的异质性。
•支持多重检测，可鉴定小型亚群。
•可以快速分析数以千计的细胞。
•非常适合血液样本和其他悬浮细胞。
•数据获取后，可以多次重复分析。
•流式细胞仪文件（FCS）可以归档。
可进行多种应用，包括免疫分型、细胞周期分析、凋亡检测（如膜联蛋白V染色检测实验）、CellEvent Caspase-3/7检测、TUNEL检测、细胞活性检测、增殖检测（如CellTrace 检测和Click-iT EdU检测）、MitoProbe检测法测定线粒体电势、利用计数微球进行细胞计数。
We do not recommend Annexin V for post-fix labeling, since fixation inactivates the function of the translocase; fixed samples would show mostly uniform labeling with Annexin V. The only options you have for apoptosis assays after fixation are to use an anti-caspase antibody or perform a TUNEL assay, such as with the Click-iT TUNEL Imaging kits.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
We have not validated the use of Click‐iT TUNEL assay for flow cytometry. Theoretically, any Click‐iT TUNEL assay for imaging can be adapted to be used with flow cytometry. In general, follow the protocol as provided but spin down the suspension cells after every step. Start with about 10∧6 cells at about 10∧7 cell/mL. Please note that flow cytometry is more sensitive than fluorescence imaging, so you should use between 1/5th to 1/10th of the azide dye detection reagent in the click reaction. All other concentrations of the click reaction reagents should stay the same. We recommend using the Click‐iT Plus TUNEL assays (C10617, C10618, C10619), as the detection reagent is provided in a separate vial, enabling you to modify the concentration used.
The Click‐iT Plus TUNEL assay protocol can be found on the following link.
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. 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.
Yes, additional Terminal Deoxynucleotidyl Transferase (rTdT) can be purchased as Cat. No. 10533-065 or 10533-075. Additional TdT reaction buffer can be purchased as Cat. No. 16314-015.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
We have not validated the use of EdU TUNEL for apoptosis detection 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.
Most tissue samples will be adequately digested in 15 minutes. The optimal incubation time will vary depending on tissue type and thickness. We have observed that brain tissue needs longer proteinase K treatment than other tissues tested.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
We have validated the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay on 5-20 µM thick FFPE sections of mouse intestine, kidney, liver, heart, and colon.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The original Click-iT TUNEL assay kits were optimized for cell culture samples and may also be used on tissue samples. We recommend using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue. The protocol was modified to improve accessibility of the TdT enzyme into the multiple cell layers of tissue samples. In addition, we found that the original Click-iT TUNEL kit may show higher non-specific binding and punctate staining in tissues compared to the Click-iT Plus TUNEL kit. One method that works well to increase permeability for tissues is to replace the detergent permeabilization step with proteinase K digestion and then refix the sample in formaldehye. The Tissue Fixation and Permeabilization protocol in section 3 of the Click-iT Plus TUNEL kit manual can be followed for tissue samples using the original Click-iT TUNEL assay. Pepsin and other proteolytic enzymes can also be used to improve permeability.
Find additional tips, troubleshooting help, and resources within our Cell Analysis 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.
-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.
Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.