Endothelial cell isolation kit

Primary cerebral ECs were isolated from newborn SHRSP rat brains (1–3 days old) by enzymatic and mechanic digestions and subsequent positive selection using microbeads magnetically labeled with CD31 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). For the enzymatic and mechanic digestions, neural tissue from neonatal brains was dissociated into single-cell suspension by using the Neural Tissue Dissociation Kit (Miltenyi Biotec) and the gentle MACS Dissociator (Miltenyi) with a specific program for neonatal brain. Afterword, the positive selection of ECs with CD31 antibody (Endothelial Cell Isolation Kit, Miltenyi) was performed by a separation over a MACS column placed in the magnetic field of a MACS separator (Miltenyi). ECs were grown in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% FBS (Euroclone Srl, Pero, Italy) and ECGS (Sigma Aldrich–Merck, Darmstadt, Germany) on gelatin-precoated dishes at 37 °C and 5% CO₂ in an humified incubator. Cells were used between passages 1–4 for all experiments, as previously reported [9 (link)]. Animal experiments for EC isolation were performed in accordance with the European Commission guidelines (Dlg 2010/63/EU) and the protocol was approved by Italian Ministry of Health (protocol n.: 448/2022-PR).