Histopaque 1083 solution

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using HISTOPAQUE-1083 solution (SIGMA, Madrid). Monocytes were purified based on their adherence to plastic in serum-free RPMI medium. Then, after 90 min, the non-adherent cells were removed by several washes with warm PBS, and the total RNA from the adherent monocytes was extracted using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, Barcelona, Spain) and purified using RNeasy microkits (Qiagen). The quality of total RNA isolated was determined using a Agilent 2100 Bioanalyser. The RNA Integrity Number (RIN) of RNA ranged from 9.10-10. Total RNA (150 ng) were amplified, labelled and hybridised to Rat Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA) using the Ambion WT Expression kit (Life Technologies) according to manufacturer's instructions.

Rats in each group were sacrificed by exsanguination, and BM was then aspirated from bilateral femurs and tibias of rats aseptically. Using density gradient centrifugation Histopaque®-1083 solution (Sigma-Aldrich, MS, USA), mononuclear cells (MNCs) were isolated from BM. To produce EPCs, BM-isolated MNCs were then resuspended in EGM-2 MV medium (Lonza, MD, USA), seeded into fibronectin (5 μg/cm²) coated six-well plates at a density of 3 × 10⁶/cm² and maintained at a 37°C incubator for 8 days. EGM-2 MV medium was replaced every two days.