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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