Enrichment of CSCs was achieved via serial trypsin treatment, as previously described [36 (link),37 (link)]. Briefly, cells at 60–80% of confluence were washed with PBS and treated with 0.05% trypsin for 2 min at 37 °C. Detached cells were plated and allowed to attach for 24 h. Then, the above procedure was repeated, and the cells obtained were considered CSCs. After the first trypsin treatment, remaining cells attached to the dishes were washed twice with PBS and incubated with 0.05% trypsin for 4 min at 37 °C. Cells detached from this trypsinization were discarded. Dishes with remaining trypsin-resistant cells were considered non-CSCs
Once isolated, cell surface marker levels of CSCs were determined with human anti-CD44-PE, anti-CD326-FITC, and anti-CD133-APC antibodies (Biolegend, San Diego, CA, USA). Samples were measured and analyzed by flow cytometry on a FACS Aria IIIu (BD Biosciences, San Jose, CA, USA).
Once isolated, cell surface marker levels of CSCs were determined with human anti-CD44-PE, anti-CD326-FITC, and anti-CD133-APC antibodies (Biolegend, San Diego, CA, USA). Samples were measured and analyzed by flow cytometry on a FACS Aria IIIu (BD Biosciences, San Jose, CA, USA).
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