Background: Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry.
Methods: To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44(+)CD24(-) CSCs subpopulation. The potential of sorted CD44(+)CD24(-) cells from different digestion to form mammosphere in culture was also compared.
Results: The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44(+)CD24(-) CSCs subpopulation cleaved into CD44(+)CD24(-) non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44(-)CD24(-) subpopulation. This cleavage effect is especially serious when cells are digested at 37°C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention.
Conclusion: Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.