Methylcellulose colony-forming unit (CFU) assay

Peripheral blood (PB)-mobilized CD34⁺ cells were cocultured for 24 hours with or without CAR CIK cells (E:T, 1:1). Afterward, 1000 CD34⁺ cells were sorted on a fluorescence-activated cell sorter Aria II, resuspended in 3 mL MethoCult H4435, and 1 mL duplicates plated in 6-well SmartDish at 5 × 10² cells per well. Plates were incubated at 37°C and 5% CO₂ for 14 days, after which different subtypes of CFUs were counted using an automated StemVision machine (STEMCELL Technologies).