Pe anti mouse cd31

Mouse adipose-derived stem cells (ASCs) were isolated from inguinal subcutaneous fat of C57BL/6 mice that were fed normal chow. The cells were cultured as described previously [12 (link)]. Briefly, subcutaneous adipose tissues were digested with collagenase type 1 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (phosphate-buffered saline) by incubation in a shaker at 37 °C for 30 min. The digestion was terminated by the addition of 10% fetal bovine serum (FBS), and centrifuged at 1200 rpm for 5 min. Cells were suspended in complete medium made of DMEM/F12 (Dulbecco modified Eagle medium) medium supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured in 37 °C at 5% CO₂ incubator. ASCs at passage 3–5 were used in all the experiments.
ASCs were identified by FACS as described previously [12 (link)] with fluorescence-conjugated anti-mouse antibodies, FITC Hamster Anti-Rat CD29 (BD Pharmingen; Cat.555005), Alexa Fluor 647 Rat anti-Mouse CD34 Clone RAM34 (RUO) (BD Pharmingen; Cat.560233), APC Rat Anti-Mouse CD90.2 Clone 53-2.1 (RUO) (BD Pharmingen; Cat.561974), APC anti-mouse CD105 (Biolegend; Cat.120413), PE anti-mouse CD31 (Biolegend; Cat.102407), or PE/Cy7 anti-mouse CD45 (Biolegend; Cat.103113), according to the manufacturer’s instructions. Flow cytometry was conducted on a Becton-Dickinson LSR I analyzer.

Left ventricular tissue was minced and digested in digestion solution containing dispase II (Wako: 383–02281) (50 μg/mL) and collagenase type 1 (Wako: 035–17604) (1 mg/mL). Digests were further dissociated with 18 G needle. Remaining debris was removed and the supernatant was filtered through 40-μm cell strainer. Cells were suspended in PBS containing 4% FBS and incubated with rat monoclonal antibody against CD31 (PECAM1) (BD Biosciences: 553370). After washing with PBS with 0.5% BSA and 2 mM EDTA, cells were incubated with anti-rat IgG microbeads (Miltenyi Biotec: 130–048), and separated with separation columns (Miltenyi Biotec: 130-042-201). For flow cytometric analysis, cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen: L34957), Tru Stain fcX (BioLegend 101320), and PE anti-mouse CD31 (BioLegend: 102408). After washing, cells were analyzed using Attune^® NxT acoustic focusing cytometer (Life Technologies). The data were analyzed by Flo Jo software (Tree Star). Isolation of adult cardiomyocytes using langendorff perfusion apparatus was performed as previously reported39 (link).