Flow Cytometric Isolation of CD44hi EpCAM Cell Subsets

Single-cell suspensions generated in PBS supplemented with 1% FBS were incubated with anti-EpCAM-FITC (1.20, Genetex, GTX30708), and anti-CD44-APC (1.20, BD Pharmingen, 559250) antibodies for 30 min on ice and analyzed on a FACSAria III Cell Sorter (BD Biosciences). CD44^hiEpCAM^hiand CD44^hiEpCAM^lo HCT116 and SW480 cells were sorted and cultured in humidified atmosphere at 37°C with 5% CO₂ for 3–5 days before collecting RNA or protein, as previously described (Sacchetti et al., 2021 (link)). The subpopulation of cells mapping in between the CD44^hiEpCAM^hi and CD44^hiEpCAM^lo gates was labelled as intermediate and was further not employed for analysis.