Pe cy7 anti mouse cd31

For qPCR validations, interstitial cells were isolated as described above, from the whole ventricles of control mice or scar area and distal area in injured mice. In experiments with Coll1a1^eGFP mice, GFP⁺DRAQ5⁺PI^- cells were sorted on Aria II with 130 μm nozzle. In absence of a fluorescent reporter (129, B6J mice), cells were first resuspended in blocking solution containing anti-mouse CD16/CD32 (1:600, Leinco Technologies, C381, clone 2.4G2) in 2% FCS 5min at RT, then stained with APC/Cyanine7 anti-mouse CD45 (1:660, BioLegend, 103115, clone 30-F11), PE-Cy7 anti-mouse CD31 (1:800, BD Biosciences, 561410, clone 390) on ice for 15min. After washing in PBS, cells were resuspended in 2%FCS+2mM EDTA and DAPI (50ng/ml) before sorting on the AriaII for DAPI^-CD45^-CD31^-cells.

AGM regions were dissected as described before.³³ (link) Tissues were incubated with collagenase I (Sigma) in phosphate-buffered saline (PBS) supplemented with 5 IU/mL penicillin, 5 μg/mL streptomycin, and 10% fetal calf serum (FCS) for 45 minutes at 37°C. The cells were stained using the following antibodies: PE-Cy7 anti-mouse CD31, APC rat anti-mouse CD117 (cKit, BD Bioscience), FITC anti-mouse CD41 (Biolegend), PE rat anti-mouse CD43 (BD Bioscience), and Alexa Fluor-700 rat anti-mouse CD45 (BD Bioscience). All antibody incubations were performed in PBS + 10% FCS for 30 minutes on ice. After washing with PBS + 10% FCS at 1000 revolutions per minute for 10 minutes, cell pellets were resuspended in 1:1000 4′,6-diamidino-2-phenylindole in PBS + 10% FCS for live/dead cell discrimination. Fluorescence-activated cell sorted events were recorded, and the cells were sorted using a FACSAria III (BD Biosciences). The results were analyzed and visualized using the FlowJo 7.6.5 software.