Cd31 antibody

Tumor tissues harvested from B20-sensitive or B20-resistant groups were minced and digested with elastase, collagenase A, and DNase 1 at 37 °C for 90 min to obtain a single-cell suspension. Platelets and RBCs were removed by performing Percoll separation, and tumor cells were sorted by FACS by using CD326 antibody (Miltenyi Biotec, Auburn, CA) as a positive selection marker for tumor cells. Endothelial cells were sorted by FACS using CD31 antibody (Miltenyi Biotec) as a positive selection marker for endothelial cells, according to the manufacture guidance (https://www.miltenyibiotec.com/US-en/applications/all-protocols/isolation-and-cultivation-of-endothelial-cells-from-adult-mouse-brain.html#gref).

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).