Annexin V binding assay has been combined with the detection of caspases activation using a FLICA assay with poly-caspase-labeled inhibitor FAM-VAD-FMK as the detector of activated caspases and Cy5-conjugated annexin V (Pozarowski et al., Cytometry 55A, pp 50-60, 2003, see Fig. 4, p. 55). Interestingly, while essentially all annexin V positive cells were also FAM-VAD-FMK positive, there a significant number of FAM-VAD-FMK positive cells that were annexin V negative. This would indicate that the time-window of apoptosis when it is detected by the FLICA assay is wider and covers earlier phase of apoptosis than the annexin V time-window. Similarly as FAM-VAD-FMK, the caspase-3 inhibitor FAM-DEVD-FMK can be used to correlate caspase-3 activation with annexin V binding. The alternative approach may be to use annexin V - FITC first, fix the cells in formaldehyde, permeabilize with Triton X and use anti-caspase-3 Ab with secondary Ab tagged with red fluorochrome. We found that Cell Signaling anti-caspase-3 Ab when combined with Zenon technology (Alexa 633 Fab fragment; Molecular Probes) is an excellent marker of caspase-3 activation (Pozarowski et al, as above) Zbigniew Darzynkiewicz -----Original Message----- From: Melendez, Johana [mailto:MelendJ@moffitt.usf.edu] Sent: Wednesday, July 28, 2004 2:23 PM To: cyto-inbox Subject: Annexin V and caspase 3 protocol Helo Flowers: I will like to know if anybody out there can send me a protocol or a reference for Annexin V staining combined with caspase 3. I found references of people discussing results , but I have not found a protocol yet. Your help will be greatly appreciated. Johana Johana Melendez, MS Research Associate Flow Cytometry Laboratory H. Lee Moffitt Cancer Center 12902 Magnolia Dr. Tampa, Florida 33612-9497 Phone (813) 745-6610 Lab. phone (813) 972-8400 x. 2005 Fax (813) 903-7140 <<Melendez, Johana.vcf>> ###################################################################### This transmission may be confidential or protected from disclosure and is only for review and use by the intended recipient. Access by anyone else is unauthorized. Any unauthorized reader is hereby notified that any review, use, dissemination, disclosure or copying of this information, or any act or omission taken in reliance on it, is prohibited and may be unlawful. If you received this transmission in error, please notify the sender immediately. Thank you. ######################################################################Received on Fri Jul 30 13:38:00 2004
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