Quantifying Pre-B Cell Colony Formation

Bone marrow cells of Lgals3^+/+ and Lgals3^−/− mice were adjusted to 5.0 × 10⁵ cells in RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10% Fetal Bovine Serum, 2 mM glutamine, 10⁻⁵ β-mercaptoethanol, and 100 mg/mL penicillin and streptomycin and maintained in 25 cm² tissue culture flasks at 37 °C in 5% CO₂ atmosphere. After 1 week in culture, adherent bone marrow cells were submitted to RNA extraction protocol as previously described^{41 (link)}. To methylcellulose medium enriched with IL-7 (Methocult M3630, Stem Cell Technologies, Canada), 5 × 10⁴ total bone marrow cells were homogenously distributed in the flasks and maintained at 37 °C in 5% CO₂ atmosphere for 1 week. The colonies (more than 50 cells) and clusters (less than 50 cells) of pre-B cells were quantified through inverted microscope Olympus CKX41SF (Olympus, Japan). The capture of images was performed using camera QColor-3 attached to the Q-Capture software (Olympus).