Analysis of homed CFSE⁺ cells. (A) BM, spleen, and blood were harvested after 3 and 24 hours homing and assayed for the CFC potential in a semisolid medium. The homing of hematopoietic progenitors was assayed by histogram profiles showing either the absolute number of CFCs recovered from one femur, the absolute number of CFCs recovered per spleen, or the number of CFCs obtained per mL of blood harvested from irradiated uninjected mice, CXCR4^+/+ CFSE⁺–injected mice, and CXCR4^–/– CFSE⁺–injected mice. BM and spleen histogram bars represent the mean ± SD of 4 independent experiments (3 mice/genotype/time), whereas blood histogram bar represents the mean ± SD of 2 independent experiments (3 mice/genotype/time). (B) Representative multiparametric flow cytometry analysis of CXCR4^+/+ CFSE⁺–and CXCR4^–/– CFSE⁺–homed cells in the BM after 3 hours homing. Cells have been first gated in a large morphologic gate including lymphocytes, monocytes, and granulocytes then viable cells (excluding 7AAD dye) were analyzed for lineage markers staining in order to draw a Lin^– gate. The percentage of Lin^– cells was similar between CXCR4^+/+- and CXCR4^–/–-injected animals and represented about 10% of viable cells. After that, Lin^– gate served for the further analysis as shown in the figure. Percentages are shown and represented means of 4 independent experiments. The differences observed for Lin^–CFSE⁺ and Lin^–Sca-1⁺c-Kit⁺CFSE⁺ cell populations between CXCR4^+/+- and CXCR4^–/–-injected mice were statistically significant (P = .019 and P = .006, respectively). (C) BM and spleen were harvested after 3 hours' homing, and multiparametric flow cytometry analyses were performed in order to study CXCR4^–/– populations that exhibited homing deficiency. Results are represented as percentage of control homed CXCR4^+/+ cells in each organ of 2 independent experiments. *P < .01.