35 mm culture dishes

Single-cell suspensions from the spleen and BM were prepared at 1 × 10⁶ and 2 × 10⁵ cells/mL, respectively, in RPMI 1640, supplemented with 10%, penicillin (100 U/mL), and streptomycin (100 mg/mL) (Invitrogen, USA) and 0.3 mL was added into 3 mL of MethoCult 03434 (StemCell Technologies, USA). The cell suspension was vortexed, allowed to stand for 10 min, and dispensed into 35 mm culture dishes (StemCell Technologies, USA) using a 16-gauge blunt-end needle and a 3 mL syringe, 1.1 mL per dish in duplicate. The cultures were incubated at 37°C, 5% CO₂ in air, and 95% humidity for 8 days after which Colony-Forming Unit Macrophages (CFU-M) were identified and counted.

Mouse CFU-E assays was performed according to manufacturer’s instructions (STEMCELL Technologies Technical Manual for Mouse Colony-Forming Unit Assays using MethoCult version 3.4.0). Bone marrow cells (4 × 10⁵) in IMDM with 2% FBS were transferred into 4 mL MethoCult M3334 methylcellulose-based medium containing EPO (STEMCELL Technologies). Samples were vortexed thoroughly and transferred in triplicate (1 × 10⁵ cells) to treated 35-mm culture dishes (STEMCELL Technologies). CFU-E colonies were enumerated after 72 h using an inverted microscope. CFU-E colony counts are reported as number of colonies/10⁶ bone marrow cells.

The CFU assay was carried out following the manufacturer’s instructions. BMNCs were adjusted to 2 × 10⁵ cells/mL, and 0.3 mL of the cell suspension was added to 3 mL of MethoCult medium and violently vortexed. Duplicated 1.1 mL aliquots of the mixture were then dispensed into 35-mm culture dishes (Stem Cell Technologies) with a 16-gauge blunt-end needle (Stem Cell Technologies) and incubated in a humidified incubator at 37°C and 5% CO₂. On the 9th to 11th day of culture, burst-forming unit-erythroid (BFU-E, early erythroid population) and colony-forming unit granulocyte-monocyte (CFU-GM) cells were identified and counted.