Flow Cytometry

Cells (1 x 10⁵) were seeded in 12-well plates. After treatment, cells were washed in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), detached from the plate using Accutase and transferred to FACS tubes. 4% formalin was used for fixation, followed by washing and cells were incubated with primary antibody, Rabbit monoclonal anti-VEGFR1 antibody (clone Y103, Abcam, 1:100) for 30 min on ice. After incubation of the secondary antibody, Alexa Flour 488-conjugated goat anti-rabbit IgG (Thermo Fisher), cells were resuspended in 200 µl PBS/1% BSA and measured on a BD FACS Canto II flow cytometer (BD Bioscience). Data was analyzed with BD FACSDiva software.