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474036 Necrosis vs Apoptosis BioAssay™ Kit

Specifications
References
Brand
BioAssay™
Kit Type
Assay
Detection Method
Fluorescent
Sample Matrix
Cell culture
EU Commodity Code
38220000
Shipping Temp
Blue Ice
Storage Temp
4°C/-20°C

Sample Type
Cell culture
Target
FAM-VAD-FMK, 7-AAD
Excitation/Emission
FAM-FLICA – 492nm/529nm 7-AAD – 546nm/647nm
Method of Analysis
Flow Cytometry, Fluorescence Microscope
Intended Use
Necrosis vs Apoptosis BioAssay™ kit simultaneously detects both apoptosis associated cytotoxicity events as well as cell death due to necrosis. This simple and straightforward tool allows researchers to understand the overall health-status of their cell populations. In addition, this kit is useful to researchers investigating the effect of their novel drug or therapeutic as it allows them to assess their experimental outcome and evaluate the overall treatment of the cells. Analyze the fluorescent signal using fluorescence microscopy or flow cytometry.
Test Principle
Assessing potential cytotoxicity properties of chemical and biological agents is a mandatory requirement for the safe distribution of pharmaceuticals, vaccines, or additives associated with food product formulations.
Necrosis vs Apoptosis BioAssay™ kit simultaneously detects both apoptosis associated cytotoxicity events as well as cell death due to necrosis. Apoptotic cells are identified using FLICA reagent probe. The FAM-FLICA probe covalently binds to active caspase enzymes, which are up-regulated during apoptosis, thus clearly labeling apoptotic cells for subsequent analysis. Non-apoptotic cells will not contain the active caspase enzymes required for FAM-FLICA to remain covalently bound within the cell structure.
Loss of the integrity of the cell membrane, indicative of necrosis or late stage apoptosis, is detected using the vital staining dye, 7-aminoactinomycin D (7-AAD), a red fluorescing live/dead stain. This dye easily penetrates cell membrane-compromised cells, binding tightly to GC rich regions of the DNA. Because 7-AAD alone may not detect cells in the early stages of apoptosis, it is essential to use it in combination with the green-fluorescent FAM-FLICA apoptosis detection reagent. Combining these two different types of fluorescent cell-status-indicator reagents within a single test can reveal a significant percentage of cells that are 7-AAD-negative (membrane intact live cells) and yet FAM-FLICA positive (apoptotic).
50-100 tests
FAM-FLICA Poly Caspase Reagent (FAM-VAD-FMK), 2 vials 7-Aminoactinomycin D (7-AAD) vital dye, 1x0.26mg vial 10X Apoptosis Wash Buffer, 1x60mL bottle Fixative, 1x6mL
100-200 tests
FAM-FLICA Poly Caspase Reagent (FAM-VAD-FMK), 4 vials 7-Aminoactinomycin D (7-AAD) vital dye, 2x0.26mg vials 10X Apoptosis Wash Buffer, 2x60mL bottles Fixative, 1x6mL
Storage and Stability
Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
References
1. Russell, J. H. & Ley, T. J. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 20, 323-370, doi:10.1146/annurev.immunol. 20.100201.131730 (2002).|2. Brunner, K. T., Mauel, J., Cerottini, J. C. & Chapuis, B. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 14, 181-196 (1968).|3. Jerome, K. R., Sloan, D. D. & Aubert, M. Measurement of CTL-induced cytotoxicity: the caspase 3 assay. Apoptosis. 8, 563-571, doi:10.1023/A:1026123223387 (2003).|4. Bedner, E., Smolewski, P., Amstad, P. & Darzynkiewicz, Z. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res. 259, 308-313,|doi:10.1006/excr.2000.4955 (2000).|5. Ekert, P. G., Silke, J. & Vaux, D. L. Caspase inhibitors. Cell Death Differ. 6, 1081-1086, doi:10.1038/sj.cdd.4400594 (1999).|6. Darzynkiewicz, Z., Bedner, E., Smolewski, P., Lee, B. W. & Johnson, G. L. Detection of caspases activation in situ by fluorochrome-labeled inhibitors of caspases (FLICA). Methods Mol Biol. 203, 289-299, doi:10.1385/1-59259-179-5:289 (2002).|7. Smolewski, P., Grabarek, J., Halicka, H. D. & Darzynkiewicz, Z. Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis. J Immunol Methods. 265, 111-121 (2002).|8. Amstad, P. A. et al. Detection of caspase activation in situ by|fluorochrome-labeled caspase inhibitors. Biotechniques. 31, 608-610, 612, 614, passim (2001).|9. Grabarek, J., Amstad, P. & Darzynkiewicz, Z. Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases. Hum Cell. 15, 1-12 (2002).|10. Murata, S., Herman, P. & Lakowicz, J. R. Texture analysis of fluorescence lifetime images of nuclear DNA with eff ect of fluorescence resonance energy transfer. Cytometry. 43, 94-100 (2001).|11. Gill, J. E., Jotz, M. M., Young, S. G., Modest, E. J. & Sengupta, S. K. 7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties. J Histochem Cytochem. 23, 793-799 (1975).|12. Lecoeur, H., Ledru, E., Prevost, M. C. & Gougeon, M. L. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods. J Immunol Methods. 209, 111-123 (1997).|13. Madhavarao, M. S., Chaykovsky, M. & Sengupta, S. K. N7-Substituted 7-aminoactinomycin D analogues. Synthesis and biological properties. J Med Chem. 21, 958-961 (1978).|14. Sengupta, S. K., Tinter, S. K., Lazarus, H., Brown, B. L. & Modest, E. J. 7-substituted actinomycin D analogs. Chemical and growth-inhibitory studies. J Med Chem. 18, 1175-1180 (1975).|15. Lee, B. W., Olin, M. R., Johnson, G. L. & Griff in, R. J. In vitro and in vivo apoptosis detection using membrane permeant fluorescent-labeled inhibitors of caspases. Methods Mol Biol. 414, 109-135 (2008).
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