Pe anti human cd31

Cells suspended in Recommended Medium were labeled with antibodies at 4°C for 30 min. Antibodies used were PE-Cy™7 Mouse Anti-Human CD34 (BD), PE anti-human CD43 (BioLegend, USA), and PE anti-human CD31 (BioLegend). After staining, cells were analyzed by Cytomics™FC 500 (Beckman, USA) with FlowJo software (Tree Star, USA).

After the treatment with MSC CM and respective control, HOVEC cells were collected and analyzed for their proliferative enhancement and expression of angiogenesis marker. Following antibodies Ki 67, PE anti-human CD31, PE/ Cy7 anti-human CD144 (VE-Cadherin), PE anti-human CD309 (VEGF-R2), Alexa Fluor 647 anti-human CD202b (Tie2/Tec), PE anti-human CD105 (Endoglin) (BioLegend, San Diego, CA, USA) were used for analysis by FACS. In brief, after the treatment, cell pellets were harvested, fixed overnight then 50μl of cell suspension was incubated with 1ul of the antibody for 25 min at 4°C in 50 μl of PBS. For analysis 10 000 cells were analyzed on a FACScan (Becton Dickinson).

Dissociated H9 human pluripotent stem cell-derived cells were resuspended in differentiation media containing diluted antibodies (dilutions listed below) for 30 min on ice. Cells were washed with differentiation media and resuspended in differentiation media + DAPI (1.35μg/ml, Biolegend) for FACS (BD FACSAria). Human pluripotent stem cell-derived cardiac myocytes used for IF-FISH were sorted by gating for SIRPA+ (PE-Cy7 anti-human CD172a/b, Biolegend, 1:200) and CD90- (APC anti-human CD90 (Thy1) Antibody, Biolegend, 1:200) cells. PSC-derived endothelial cells were sorted by gating for CD31+ (PE anti-human CD31, Biolegend, 1:200) cells.