Neonatal cardiac endothelial cell isolation kit

Primary Sca1⁺Bmi1⁺CD45⁻ and PDGFRα⁺ mesenchymal-like cells were sorted after Langendorff digestion and expanded in Iscove's modified Dulbecco's medium (IMDM, ThermoFisher, 12440053) containing 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin and 2 mM l-glutamine (all from Invitrogen), 10³ units ESGRO Supplement (Millipore, ESG1106), 10 ng/ml EGF (epidermal growth factor; Sigma, SRP3196) and 20 ng/ml FGF (fibroblast growth factor; Peprotech, 100-18B) (37 °C, 3% O₂, 5% CO₂). Primary adult cardiac endothelial cells (CD31⁺) were obtained with the Neonatal Cardiac Endothelial Cell Isolation Kit (Miltenyi, 130-104-183). CD31⁺ primary cells and the 1g11 endothelial cell line [39 (link)] were expanded in VascuLife VEGF Endothelial Medium Complete Kit (Lifeline Cell Technology, LL-0003) (37 °C, 21% O₂, 5% CO₂). Primary cardiac cells were used for the experiments at passage ≤9.

Endothelial cells were isolated from fresh kidneys that were extracted and digested in an endothelial cell digestion buffer: MCDB-131 complete media, collagenase A (1 mg/mL), collagenase B (1 mg/mL), and DNase1 (100 μg/mL) at 37 °C for 20 min. Cell suspension was subjected to cell sorting using the neonatal cardiac endothelial cell isolation kit (130-104-183, Miltenyi Biotec) and autoMACS Magnetic Cell Separator of Miltenyi according to the manufacturer's instructions.