S1300ex

For quantification of EPCs in the BM, circulation, and retina, fluorescence‐activated cells sorter analysis (FACS) was used as described 36. Samples were transferred into 12 mm × 75 mm polystyrene round‐bottom tubes and incubated with CD45‐FITC (no. 103108; Biolegend, San Diego, CA), CD133‐PE (no. 141204; Biolegend), CD34‐BV421 (no. 119321; Biolegend), and VEGFR2‐APC (no. 136406; Biolegend) antibodies for 1 hour in the dark. The red blood cells were lysed by adding 2 ml of red blood cell lysis buffer (BD Biosciences, Faranklin Lakes, NZ) for 15 minutes. The remaining cells were washed in 2 ml PBS containing 10% fetal bovine serum and pelleted by centrifugation at 300g for 5 minutes and finally resuspended in 250 ml paraformaldehyde (4%). The sample tubes were stored at 4°C in the dark until analysis. The samples were measured by Stratedigm S1300Ex (Stratedigm, San Jose, CA) and CD34+/CD45−/VEGFR2+/CD133+ population was analyzed by FlowJo software (Life Science Software Company, Ashland, Oregon).