Keith, Robert and Cytometry List, I agree that flow is not necessary to detect apoptosis and it is difficult to control membrane damage when removing anchorage dependent cells. I am not sure the chronology of phosphatidyl serine exposure (Annexin V positive) relative to spontaneous detachment of apoptotic cells from the surface. Regarding the caspase-3 kits that use tetrapeptide substrates that become cleaved by caspases to liberate one of the coumarins, I think you will find that you probably will need more cells than would normally be cultured in a 96 well plate to get enough detectable caspase-3 activity using that technology. Also, keep in mind that you may be detecting some caspase-7 present if the substrate is Ac-DVED-AMC. In some literature you will find the use of the term "DEVDase activity" because of the lack of specificity of the substrate. This probably doesn't make any difference if all you want to measure is caspase activity as a marker of apoptosis. Regarding the use of tetrazolium reagents such as MTT to detect apoptosis, that approach will work; but, it will depend on the time the assay is run after induction of apoptosis. In fact shortly after induction of apoptosis, you will see an increase in reduction of tetrazolium reagents, then it will fall as the cells shut off their metabolism. Caspase-3 activity (DEVDase) will appear before reduction of tetrazolium reagents has disappeared. One approach that would work for flow is to use the FITC-VAD-FMK pan-caspase inhibitor. The VAD (valine, alanine, aspartic acid) peptide binds to the active site of active caspases (i.e. only during apoptosis) and the -FMK (fluoromethyl ketone) causes binding to be irreversible. It would probably still bind to active caspases even if the cell membranes are damaged by the removal process from the plastic surface. Another option might be to use one of the marker antibodies such as Anti-PARP p85 Fragment pAb. The antibody does not detect the intact PARP molecule; but it specifically detects a neo-epitope on the large fragment of PARP that is generated as a result of caspase cleavage. Ty Lee -----Original Message----- From: Keith Bahjat <kbahjat@ufl.edu> To: cyto-inbox Date: Tuesday, May 02, 2000 2:25 PM Subject: Re: Annexin V/PI assays for adherent cell lines. > >Robert, > >While flow cytometric assays are nice, they are not always necessary. I find >flow useful because I can simultaneously measure multiple parameters at >once, like apoptosis within a specific T cell population. but in your case, >you have a purified cell population. Here, an assay such as a fluorometric >caspase 3 (kit available from Bio-Rad), or MTT (Molecular Probes) would be a >much simpler way to accomplish the same thing. And these can be done in a 96 >well plate. > >Hope this is helpful. > >Keith Bahjat >kbahjat@ufl.edu > > >on 5/1/00 7:54 AM, Robert Connelly at rconnelly@ameritech.net wrote: > >> Hello, >> We are having some problems developing an annexin V/PI assay for the >> SK-OV-3 and BG-1 both epithelial ovarian cancer cell lines. We exposing >> the cells in vitro to cisplatin for 24, and 48 hours. We are using the >> Clontech EGFP-annexin V kit with PI. >> The literature is controversial and confusing particularly in the >> harvesting of the cells from the substrate. We have been harvesting with >> Trypsin-EDTA and scraping. Unfortunately scraping increases ( compared >> to the Trypsin-EDTA group) the annexin negative/ PI positive population >> in the untreated control group. >> To date we have not seen any annexin V positive results after 24 or 48 >> hours of cisplatin exposure. Our next experiments >> we be a time course study of cisplatin exposure for PS externalization >> to occur. >> If the group could shed some light or give constructive suggestions it >> would be greatly appreciated. >> >> >> Robert Connelly >> >> Reproductive Oncology Laboratory >> >> University Hospitals of Cleveland >> >> 1-216-844-1553 >> >> >> > >
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