Quantifying Colony-Forming Potential of Bone Marrow

To estimate the number of colony-forming units (CFU-Fs) in the obtained marrow suspensions, nucleated cells were plated in triplicate at a density of 8, 0 × 10³/cm² (Corning Incorporated, New York, NY, USA) in 2 mL of α-MEM supplemented with 20% FBS. After 3 days of incubation at 37°C in 5% CO₂, the nonadherent hematopoietic cells were removed, and the medium was changed. At day 14, the colonies were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with 1% crystal violet (Sigma-Aldrich). Colonies with more than 50 cells were counted [48 (link)]. The efficiency of colony formation was expressed as the mean colony number relative to the 100000 bone marrow nucleated cells plated.