hPSCs or ITGA5 KD hPSCs were seeded in a 96-well plate at a density of 2.5 × 103 cells per well. The next day, cells were starved and activated either with or without TGF-β. After 24 hours, cells were washed with PBS and fixed with 4% formaldehyde (Sigma) for 15 min at room temperature. Later, fixed cells were blocked in serum-free medium with 1% BSA for 30 min at room temperature. The cells were incubated with 10 μM AV3-FAM in serum-free medium with 0.1% BSA for 60 min at room temperature. After incubation, the cells were washed with PBS, and nuclei were stained with DAPI followed by imaging using EVOS fluorescence microscope (Thermo Fisher Scientific).