Iscove s modified dulbecco s medium imdm

Mouse Lin⁻ BM cells were isolated and kept at −80°C overnight. Cells were thawed the following day and seeded in a 24-well plate at 1 × 10⁵ cells/well in StemSpan medium supplemented with complete growth medium supplement (CGMS; 1% penicillin/streptomycin, 50 ng/mL mouse SCF, 100 ng/mL FLT-3L, 100 ng/mL IL-11, and 20 ng/mL mouse IL-3; all from PeproTech) and incubated for 2 days. Plates coated with Retronectin (Takara Bio) were used to preload viral constructs, and the first round of transduction was completed with prestimulated Lin⁻ cells. A new Retronectin-coated plate was used for preloading viral vectors on the following day (day 0), and a second round of transduction was conducted by transferring transduced cells to the new plate. After overnight incubation, cells were transferred to 12-well plates and cultured in Iscove's modified Dulbecco's medium (IMDM; Biochrom) supplemented with CGMS. After day 15, cells were seeded at 100 cells/well in 96-well plates and cultured for an additional 2 weeks. Trypan blue exclusion counting was performed on days 1, 4, 6, 8, 11, and 15.