Isolation and Culture of Mouse lin- BMCs

Mouse lin⁻ BMCs were isolated from bone marrow as previously described [13 (link),28 (link)]. Briefly, femur and tibia bones were flushed with MEM media (Minimum Essential Medium, ThermoFisher Scientific) until translucent. Cell pellets from the flushed media were depleted of red blood cells (RBCs) using the Red Blood Cell Lysis Buffer (Millipore Sigma, Burlington, MA, USA). lin⁻ BMCs were isolated using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Bergisch Gladbach, North Rhine-Westphalia, Germany). Isolated lin⁻ BMCs were cultured on fibronectin-coated (Sigma Aldrich) plates in EBM™-2 Endothelial Basal Medium-2 (Lonza) supplemented with EGM™-2 MV Microvascular Endothelial SingleQuots^TM Kit (Lonza) containing vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), insulin like growth factor (IGF), ascorbic acid, hydrocortisone, gentamicin, amphotericin-B, and 20% fetal bovine serum (FBS). Cells at passages 3–7 were used for experimentation.