Phenotypic analysis of G-CSF/SCF-dependent colonies.

WT and Ik^L/L BM cells were cultured in methylcellulose medium supplemented with G-CSF plus SCF. (A) Representative colonies (day 7) for each genotype. Original magnification, × 80. 6 independent experiments were performed. (B) BM cells were cultured as above for the indicated number of days. At each time point, the number of cells per colony was counted for the 10 largest colonies in each plate; bars represent the means ± SDs. (C) Similar proliferation kinetics between WT and Ik^L/L cells, as revealed by decrease of the CFSE label. Left: CFSE profile of BM cells at day 0 of culture (black histograms), and of representative WT and Ik^L/L colonies after 6 days of culture (white histograms). For each genotype, 6 colonies were analyzed, all of which showed similar CFSE levels. Right: Lin⁻ BM cells were sorted, labeled with CFSE, and 10⁵ cells were put in culture. Cells from entire plates were harvested after 1 to 3 days of culture and their CFSE levels analyzed. Days of harvest are indicated next to specific histograms. (D) Analysis of apoptosis by Annexin V/propidium iodide staining. Left: isolated colonies at day 6; 6 colonies were analyzed for each genotype and displayed similar phenotypes (with the exception of a single WT colony that had approximately 10% annexinV-positive cells, not shown). Right: 10⁵ sorted Lin⁻ cells were put in culture; cells from entire plates were harvested after 2 days and analyzed. (E) For each genotype, 10 colonies were pooled and analyzed for Gr-1 and Mac-1 expression after 5, 6, and 7 days of culture. Numbers indicate the percentage of cells in each quadrant.