Percoll cell separation solution

A density gradient centrifugation approach was used to collect mononuclear cells from the rabbit bone marrow with the Percoll cell separation solution (Solarbio, China), after which these cells were cultured in T25 cell culture flasks containing αMEM supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) at 37°C in a humidified 5% CO₂ incubator. Media were changed every 3 days, and rBMSCs from passages 3–5 were used for all experiments. The surface phenotype of these rBMSCs was assessed via flow cytometry by staining 5 × 10⁵ BMSCs from passage 3 with antibodies specific for CD34 (1 : 1000; BioLegend, CA, USA), CD45 (1 : 1000; BioLegend), CD90 (1 : 500; BioLegend), CD105 (1 : 1000; BioLegend), HLA-DR (1 : 1,000; Abcam, Cambridge, UK), and CD44 (1 : 1,000; Abcam) for 30 minutes at 4°C while protected from light. Cells were then rinsed with PBS and analyzed using a FACScan flow cytometry system (CytoFLEX; Beckman Coulter, USA).

Two-week-old male Beagle dogs were purchased from the Experimental Animal Center of Guangxi Medical University (Nanning, China). All animal experimental procedure was approved by the Animal Care and Use Committee of Guangxi Medical University. The isolation procedure was performed as described in previous studies [17 ]. Mononuclear cells were isolated from the bone marrow by density gradient centrifugation with Percoll cell separation solution (Solarbio, China), plated in T25 cell culture flasks coated with fibronectin (Sigma, USA) overnight and then cultured in complete endothelial growth medium-2 (EGM-2; Lonza, USA) at 37 °C in a 5%CO₂ humidified incubator. Cells were cultured until 80–90% confluent, at which time they were passaged. Following two such passages, cells were utilized for downstream experiments.