Astrios sorter

Cardiac cells were isolated from the ventricles of 2-day old neonatal rat hearts according to previously described methods [25 (link), 28 (link)]. Animals were sacrificed using humane standards approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University. Ventricles were harvested, minced, and enzymatically dissociated by serial digestion with trypsin and collagenase. Isolated cells were resuspended in DMEM/F-12 with 10% Fetal Bovine Serum and 10% Horse Serum, and pre-plated for 45 min at 37 °C to increase the fraction of cardiomyocytes in the cell suspension [28 (link), 29 (link)]. To assess cardiomyocyte purity, pre-plated cells were fixed and permeabilized according to the manufacturer instructions (BD Biosciences # 554714), then stained with cardiac troponin T (cTnT) primary antibody (Abcam ab45932) and Alexa Fluor-conjugated secondary antibody. To assess fraction of endothelial cells, pre-plated cells were washed with PBS and stained with PE-conjugated CD31 antibody (BD Biosciences #555027) and APC-conjugated CD90 antibody (BD Biosciences #561409). Stained cells were analyzed in Beckman Astrios Sorter and BD FACSCanto A analyzer at the Duke Cancer Institute’s Flow Cytometry Shared Resource. Endothelial cells and fibroblasts were identified as CD31⁺ and CD90⁺ /CD31⁻ [30 (link)] cells, respectively.