hPSCs were seeded at a density of 4 × 105 cells per T25 flask. The next day, cells were starved and activated either with or without TGF-β. After 24 hours, cells were trypsinized, and cell numbers were diluted to 1 × 105 cells/ml. Cells were incubated at 37°C for 30 min to allow receptor recovery. Then, different concentrations (1, 2.5, 5, and 10 μM) of AV3-FAM were added to the suspension cells containing 2% FBS and incubated at 4°C for an hour. Cells were then centrifuged at 300g at 4°C for 5 min. The supernatant was decanted without disturbing the pellet, and cells were washed three times with 0.5% FBS/cold PBS and then fixed in 0.5% formaldehyde for 10 min at 4°C. Cell fluorescence was measured with flow cytometry (BD FACSCalibur).