Mouse anti human cd31 antibody

Co-cultures were fixed with 3.7% formaldehyde-PBS for 20 min, treated with 0.1% TX-100-PBS for 20 min and washed twice with PBS, and incubated overnight with 63 ng/ml mouse Anti-Human CD31 antibody (BD Pharmigen, 550389) in PBS containing 1% BSA at 4° C. Following the PBS wash and samples were then incubated with 3 μg/ ml Alxea 488 goat secondary antibody in PBS for 1 h and then washed twice with PBS before staining cellular DNA with Hoechst (2 μg/ ml).

Age at death and gender of the anonymous donors of human ocular tissue were as follows: 39-year-old female; 46-year-old male; 48-year-old male; 18-year-old female; and 36-year-old male. Death to endothelial cell isolation time varied from 11 to 22.5 hours. Our method for isolation of endothelial cells from human retina and choroid has been published in detail.^{63 (link)} In brief, the retina and choroid were dissected from both posterior globes, and separately digested with 0.3 mg/ml Dispase (Thermo Fisher Scientific-GIBCO, Grand Island, NY) and 0.25- 1 mg/ml type II collagenase (Sigma-Aldrich, St Louis, MI). After 7-10 days of culture in MCDB-131 medium (Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS) (GE Healthcare Life Sciences-HyClone, Logan, UT) and endothelial growth factors (EGM-2 SingleQuots supplement, omitting FBS, hydrocortisone and gentamicin; Lonza-Clonetics, Walkersville, MD) at 37 °C, endothelial cells were purified using magnetic Dynabeads (Thermo Fisher Scientific-Invitrogen Dynal, Oslo, Norway) coated with mouse anti-human CD31 antibody (BD Pharmingen, San Diego, CA), and grown in modified MCDB-131 medium with 10% FBS. Subculturing of retinal endothelial cells was performed with 0.05% trypsin (Thermo Fisher Scientific-GIBCO). The cell isolates were used at passage 2 or 3.