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Product Details
| Cat # +Size | K402-50 |
|---|---|
| Size | 50 assays |
| Detection Method | Flow cytometry (Ex/Em = 488/520 nm for FITC, and 488/623 nm for PI) and fluorescence microscopy (FITC and rhodamine filters) |
| Species Reactivity | Mammalian |
| Applications | The TUNEL-based assay kit provides complete components including positive and negative control cells for convenient detection of DNA fragmentation in cultured cells and tissue sections. |
| Features & Benefits | • Simple procedure; takes ~ 3 hours • Fast and convenient • The assay is sensitive and stable • The TUNEL-based assay kit provides complete components including positive and negative control cells for convenient detection of DNA fragmentation in cultured cells and tissue sections. |
| Kit Components | • Positive Control Cells • Negative Control Cells • Wash Buffer • Reaction Buffer • TdT Enzymes • Rinse Buffer • Anti-BrdU-FITC Antibody • PI/RNase Staining Buffer |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Internucleosomal DNA fragmentation is a hallmark of apoptosis in mammalian cells. BioVision’s ApoDIRECT In Situ DNA Fragmentation Assay Kit provides complete components including positive and negative control cells for conveniently detecting DNA fragmentation by fluorescence microscopy or flow cytometry. The TUNEL-based detection kit utilizes terminal deoxynucleotidyl transferase (TdT) to catalyze incorporation of fluorescein-12-dUTP at the free 3’-hydroxyl ends of the fragmented DNA. The fluorescein-labeled DNA can then be observed by fluorescence microscopy or analyzed by flow cytometry.
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All my cells both treated and control is showing positive PI stain. Why so?
The reason why PI is staining all sample types is because the Reaction buffer and the Rinse buffer have a detergent in them, Triton-X. You need the detergent to allow the probe to enter the nucleus to stain the DNA. So if you use the kit, all cells should stain positive for PI, and PI is just being used as a counterstain.
Can this assay differentiate between necrosis/apoptosis?
TUNEL assay is positive for both necrotic and apoptotic cells and hence cannot distinguish. You can run DNA gel to differentiate between them- while necrotic cells will show a smear due to non-specific cleavage, apoptotic cells has activated endonucleases that cleaves the DNA into fragments of approximately 180-200bp that looks like a ladder on the DNA gel.
In the last step of the protocol (step 10), there is no description how the cells are transferred to slides for fluorescence microscopy. May I know how the cells are transferred to slides?
We take 50-100ul of cells and put it on the slide, invert a coverslip on it and seal with nail-polish. Poly-lysine treated slides are better.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
BioVisions’s assay kits expire 6 months from the date of shipment. Some components of the kit may expire sooner as mentioned in the data sheet
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should the Control cells be fixed? Also, my Control cells are not pelleting after Step IV-B-2. What should I do?
Control cells are already fixed and therefore should not be fixed again. Please centrifuge at a higher speed for a longer time to pellet them. Sometimes fixed cells required higher centrifugation speed and longer duration to form a pellet.
Could you please provide some information on how to do the gating for flow cytometry?
For flow cytometry, we recommend a standard dual parameter DNA doublet discrimination display with the DNA Area signal on the Y-axis and the DNA Width (Becton-Dickinson) or DNA Integral (Coulter) signal on the X-axis. From this display, a region is drawn around the non-clumped cells and the second gated dual parameter display is generated. The normal convention of this display is to put DNA (Linear Red Fluorescence) on the X-axis and the d-UTP (Log Green Fluorescence) on the Y-axis. By using the dual parameter display method, not only are apoptotic cells resolved better, but you can also determine the stage of cell cycle they are in.
Amir Apelbaum et al., Type I Interferons Induce Apoptosis by Balancing cFLIP and Caspase-8 Independent of Death Ligands. Mol. Cell. Biol., Feb 2013; 33: 800 - 814.
Goplen, D. et al.. {alpha}B-Crystallin Is Elevated in Highly Infiltrative Apoptosis-Resistant Glioblastoma Cells. Am J Pathol. 2010; Oct 2010; 177: 1618 - 1628.
Bahl, K. et al. Analysis of Apoptosis of Memory T Cells and Dendritic Cells during the Early Stages of Viral Infection or Exposure to Toll-Like Receptor Agonists. J. Virol. 2010 84: 4866-4877.
Suwan K et al (2009) J. Biol. Chem.; 10.1074/jbc.M806927200.
Vogt KSC ET AL (2008) j. Exp. Biol. 211: 731-740.
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