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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