After the treatments, the cells were washed with 10 mL phosphate buffered saline, and 3 mL trypLE (Gibco 12604-013; Thermo Fisher Scientific, Waltham, MA, USA) was added to each flask to disassociate the cells from the flask. The activity of trypLE was stopped by adding 3 mL trypsin neutralization solution (Gibco R002100), which does not contain serum. The cells were counted after resuspension and collected by centrifugation. All cell pellets were resuspended in stain buffer (BD Biosciences, San Jose, CA, USA) at 4×106 cells/mL or 105 cells/25 μL.
Of the cells in stain buffer, 25 μL were transferred to flow cytometry-compatible polystyrene round-bottom tubes (BD Biosciences 352008), and 10 μL allophycocyanin- or phycoerythrin (PE)-labeled antibodies (anti-human VEGFR1, VEGFR2, VEGFR3, PDGFR-β, Tie-2, or CD31 from R&D Systems, Inc., Minneapolis, MN, USA) were added to the appropriate tubes. The samples were incubated in the dark at 4°C for 45 minutes. Untreated cells (100 μL) were used as the no-antibody control.
Of the cells in stain buffer, 25 μL were transferred to flow cytometry-compatible polystyrene round-bottom tubes (BD Biosciences 352008), and 10 μL allophycocyanin- or phycoerythrin (PE)-labeled antibodies (anti-human VEGFR1, VEGFR2, VEGFR3, PDGFR-β, Tie-2, or CD31 from R&D Systems, Inc., Minneapolis, MN, USA) were added to the appropriate tubes. The samples were incubated in the dark at 4°C for 45 minutes. Untreated cells (100 μL) were used as the no-antibody control.