Downregulation of c-kit expression in absence of c-kit activation by use of SCF-deficient (UNC Sl⁻/Sl⁻) stroma cells. Stroma feeder was prepared by seeding 5 × 10⁴ stroma cells per well into 6-well tissue culture plates (Nunclon). TF-1 cells grown in GM-CSF–containing medium were washed three times in RPMI medium and subsequently transferred to irradiated (17,600 rad) stroma. Usually every 7 days the TF-1 cells were sequentially transferred to fresh prepared stroma. An aliquot of cells was analyzed for expression of c-kit as described in Materials and Methods. Aliquots of each transfer were confirmed to be negative for factor-independent mutant cells by recloning in medium with and without GM-CSF. Cultures that contained mutant cells were excluded.