Factors influencing percentage yield of side population isolated in ovarian cancer cell line SK-OV-3

Isolation of side population (SP) cells has been recognized as a useful technique for the isolation and identification of hematopoietic stem cells or cancer stem cells (CSCs). Thus the yield and purity of isolated SP cells would have a profound influence on the research outcomes in these two important areas. Hoechst 33342 exclusion assay technique has been used for the identification of SP cells. However, diverse Hoechst staining protocols giving different SP yields even from the same tissue type or same cell line have been reported in different laboratories. In this study we systematically investigated the underlying factors influencing the SP yield using Hoechst dye staining and a robust platform of flow cytometric analysis of the human ovarian cancer cell line SK-OV-3. Our study revealed that SP yield was not only affected by the Hoechst 33342 concentration, staining cell density, staining cell viability, staining duration, staining medium, flow cytometric setting and SP gating strategy, but was also affected by the cell passage number in SK-OV-3. This is the first systematic study on the factors affecting SP yield in adherent cells that mimic many solid tumour tissues. Our results provide important technical guidelines to help ensure reproducible and comparable results in SP and CSCs study.