Cytopeia influx cell sorter

Aldehyde dehydrogenase 1 is considered as a marker for stem cells and is present in tumors of several organs, including ovary (Deng et al., 2010 (link), Flesken-Nikitin et al., 2013 (link)). Primary COVCAR cells (n = 8 animals) obtained and maintained individually as described previously were subjected to ALDEFLUOR assay (STEMCELL Technologies, Vancouver, Canada) following the manufacturer's protocol. ALDH1 enzyme activity was measured in this assay followed by fluorescence-activated cell sorting (FACS) to separate ALDH1⁺ and ALDH1^- cells. Briefly, COVCAR cells (∼5 million cells per mL) were added to ALDEFLOUR reagent (1 μl/10⁶ cells) without or with diethylamino benzaldehyde (DEAB; an ALDH1 inhibitor added as negative control) and incubated at 37°C for 30 min. After removing the ALDEFLOUR reagent, the cells were suspended in ALDEFLUOR assay buffer and subjected to FACS. Cells with high ALDH1 activity (ALDH1⁺) and low ALDH1 activity (ALDH1^-) were sorted at 4°C in a Cytopeia Influx cell sorter (BD Biosciences, San Jose, CA) and collected into serum-free stem cell medium [SCM; X-VIVO 20 medium (Lonza, Walkersville, MD) supplemented with 5 μg/mL bovine insulin (Sigma-Aldrich), 20 ng/mL recombinant human epidermal growth factor (PeproTech, Rocky Hill, NJ), and penicillin-streptomycin-fungizone solution (Invitrogen)].