Bone marrow-derived mononuclear cells (UPECLIN HC FM Botucatu Unesp)

Bone marrow was harvested following a 3-day stimulation with granulocyte colony-stimulating factor (subcutaneous injection; 5 μg/kg). 30 mL of bone marrow (diluted 1:1 with saline) were added to 20 ml Ficoll-Hypaque Premium solution. Afterwards, the tubes were centrifuged at 300 g for 30 min at 18°C. The ring of mononuclear cells that formed above the Ficoll layer was collected. Mononuclear cells were diluted in RPMI culture medium and centrifuged at 300 g for 15 min at 18°C. After centrifugation, the precipitate was resuspended in RPMI medium. The contents of all the tubes were aggregated and then centrifuged at 300 g, at 18°C for 10 min. At the end of this step, the precipitate was resuspended to a final volume of ∼50 mL, with albumin saline solution (ASS) to obtain a final concentration of 1 ×10⁸ mononuclear cells/mL.