By Demetrius Matassov, Terri Kagan, Julie Leblanc (auth.), Hugh J. M. Brady (eds.)
A set of state-of-the-art innovations for detecting and quantifying apoptosis, figuring out its biochemistry, and for deciding on the genes and proteins that control and hold it out. defined in step by step element, those quite simply reproducible tools diversity from circulate cytometry and immunohistochemical systems to kinase job assays, yeast two-hybrid screening, and the cloning of novel genes by means of differential expression. The protocols stick to the winning equipment in Molecular Biology™ sequence structure, each delivering step by step laboratory directions, an creation outlining the main in the back of the method, lists of kit and reagents, and pointers on troubleshooting and fending off recognized pitfalls. Apoptosis equipment and Protocols constitutes a key technical connection with the numerous methodologies utilized in the sphere, in addition to supplying amateur and skilled researchers alike robust instruments to light up the phenomenon of programmed phone loss of life.
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5. 5 Duke, R. , and Young, J. D. -E. (1996) Cell suicide in health and disease. Sci. Am. 275, 80–87. 6. 6 Greenberg, A. H. (1996) Granzyme B-induced apoptosis. Adv. Exp. Med. Biol. 406, 219–228. 7. , and Saito, T. (1995) Fas-mediated cytotoxicity by freshly isolated natural killer cells. J. Exp. Med. 181, 1235–1238. 8. 8 Eischen, C. M. and Leibson, P. J. (1997) Role for NK-cell-associated Fas ligand in cell-mediated cytotoxicity and apoptosis. Res. Immunol. 148, 164–169. 9. 9 Duke, R. , and Cohen, J.
And 4 µg/mL 7AAD to the modified staining buffer. 5. DiOc6 Stock 1. 4 µM DiOc6(3) stock: DiOc6(3) is from Molecular Probes. Prepare in ethanol (analytical grade) in 1-mL quantities. Store at 4°C in the dark. 6. Buffer for Annexin V Assay 1. FITC or PE-labeled annexin V can be obtained from Pharmingen. 2. 8 mM CaCl2. 3. 1. Light Scatter Analysis 1. 1% w/v NaN3. Analyze FSC vs SSC, both on linear scales, of cells using a flow cytometer. 2. Set a gate for viable cells on the FSC vs SSC dot plot, by analyzing control viable cells (region 1, Fig.
A. (1999) Prodomains-adaptors-oligomerization: the pursuit of caspase activation in apoptosis. Trends Biochem. Sci. 24, 1–4. 12. Kumar, S. (1999) Mechanisms of caspase activation in cell death. 12 Cell Death Differ. 6, 1060–1066. 13. 13 Thornberry, N. , Rano, T. , Peterson, E. , Rasper, D. , et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17,907–17,911.