• Non-cytotoxic assay arrests further apoptotic activity via caspase inhibition.
• Cell permeablity permits direct visualization of cytosolic apoptotic events.
• Apoptotic cell population does not diminish over time.
• Add reagent directly to cells. No special buffer or media needed. No preparation of cell lysates required. Simple wash procedure.
• Works in diverse cell lines: human, rodent, Drosophila.
• Can be performed in conjunction with Annexin staining, TUNEL, antibody staining, or with other APO LOGIX reagents on the same population of cells.
• Permits high through-put screening. Protocol can be adapted for ex vivo as well as in situ experiments.
• Applications - Works with fluorescence microscope, 96-well fluorescence plate readers
• Yields both quantitative and qualitative results. Gives strong signal with little background noise.

APO LOGIX SR kits contain a generic sulforhodamine labeled caspase inhibitor (sulforhodamine-peptide-fluoromethyl ketone). This reagent is cell permeable and is used on whole cells to detect apoptosis. Apoptotic cells are detected by a fluorescence plate reader or fluorescence microscope using an excitation source at 550nm and measuring emission at 595nm. The assay takes about 1 hr to complete

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2. Walker, N. P., R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett and L. D. Hammill. 1994. Crystal Structure of the Cysteine Protease Interleukin-1ß-Converting Enzyme: A (p20/p10)2 Homodimer. Cell 78:343-352.
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4. Rotonda, J., D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J. W. Becker. 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3(7): 619-625.
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7. Amstad, P.A., G.L. Johnson, B.W. Lee and S. Dhawan. 2000. An in situ marker for the detection of activated caspases. Biotechnology Laboratory 18: 52-56.
8. Bedner, E., P. Smolewski, P.A. Amstad and Z. Darzynkiewicz. 2000. Activation of caspases measured in situ by binding or fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Research 259: 308-313.
9. Smolewski, P., E. Bedner, L. Du, T.-C. Hsieh, J. Wu, J. D. Phelps and Z. Darzynkiewicz. 2001. Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44: 73-82.
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11. Carcia-Calvo, M., E. Peterson, B. Leiting, R. Ruel, D. Nicholson and N. Thornberry. 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273: 32608-32613.
12. Hirata, H., A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okazaki, K. Yamamoto and M. Sasada. 1998. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187: 587-600

• Lyophilized SR-VAD-FMK
• 10X Wash Buffer
• 10X fixative