Pe conjugated cd31 antibody

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.

For Tie2Cre;Piezo2^cKO and control mice, lungs were removed from adult mice (n = 4) and were processed into a single-cell suspension. Cells were then stained with PE-conjugated CD31 antibody (BD Pharmingen) for 30 min to 1 h on ice and were subjected to fluorescence-activated cell sorting (FACS). DAPI⁺ cells were excluded, and DAPI⁻ CD31⁺ and DAPI⁻ CD31⁻ cells were sorted into separate tubes containing TRIzol. Total RNA was isolated for qRT–PCR. For Wnt1Cre;Piezo2^cKO and control pups, DRG were removed from newborn pups (n = 5) and placed into TRIzol. Total RNA was isolated and subjected to qRT–PCR as previously described^{4 (link)}.