Identification and validation of side population cells from ovarian cancer cell lines.

<p>(A) Identification of SP cells in established human ovarian cancer cell lines. SKOV3 and A224 cell lines were labeled with Hoechst 33342 dye and analyzed by flow cytometry. The SP cells, which disappeared in the presence of Verapamil (a multidrug transporter inhibitor), are outlined and shown as the percentage of the total cell population. Similar results were obtained for three independent measurements. (B) Validation of randomly selected genes from the SP gene list on the SP and MP cells isolated from the SKOV3 cell lines. (C, D) Colony forming efficiency assay: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines. For the analysis of colony forming efficiency (CFE), SP and MP cells were sorted and plated in equal numbers in tissue culture six well plates and grown for 14 days. The cells were then fixed with cold methanol and stained with 0.5% crystal violet solution to count the number of colonies by microscopy. The experiments were carried out in triplicates. (E, F) Passage Number: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines were evaluated as a function of passage number. Cells were fixed with cold methanol and stained with 0.5% crystal violet.