2.3. Flow Cytometry Analysis

Cells were collected and washed twice in buffer solution (PBS containing 2% FBS). For the cell surface markers, the cells were incubated with fluorescein-isothiocyanate- (FITC-) conjugated or phycoerythrin- (PE-) conjugated primary antibodies at 4°C and protected from light. For the intracellular biomarkers, cells were fixed in 4% paraformaldehyde (PFA) for 20 min and washed with the buffer solution, then pretreated with 0.5% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and washed with the buffer solution again. For indirect staining, the cells were incubated with unconjugated primary antibodies at 4°C followed by incubation with FITC- or PE-conjugated secondary antibodies at 4°C with protection from light. Finally, samples were washed and kept in the dark prior to analysis. Single staining flow cytometry was performed using EPICS XL (Beckman Coulter, Kraemer Boulevard Brea, CA, USA) with EXPO032 ADC XL 4 Color software (Beckman Coulter). Double staining flow cytometry was performed using BD FACSCalibur (BD) with CellQuest software (BD). The primary antibodies used are detailed in the Supplementary Material (Table S1).