6–8 weeks-old male and female +/+ and +/w mice were sacrificed, and their thymi were harvested. A) Cell-surface CXCR4 expression on TCRβ+ SP thymocytes from the mouse genotypes indicated in the inset (+, wild type Cxcr4; w, WHIM allele). Bars represent mean ± SD CXCR4 median fluorescence intensity (MFI) of 3 mice (white symbols) analyzed in duplicate. Data are from one experiment representative of 4 independent experiments and analyzed by multiple t-test. B and C) CXCL12-induced chemotaxis of SP thymocytes. After processing the thymi of +/+ and +/w mice, CD4 and CD8 SP thymocytes were isolated by magnetic-activated cell sorting, and then were differentially stained with green and violet calcein AM before pooling the 4 SP populations together. Chemotactic responses of this SP pool to increasing doses of mouse CXCL12 (x-axes) were tested in transwell assays and migrated cells were counted and analyzed by FACS. B) Total number of migrating SP cells. C) Chemotaxis index for each SP subset. The Cxcr4 genotype (+, wild type; w, WHIM allele) code for A and C is indicated in the inset. In B and C, data are the mean ± SEM of 3 independent experiments analyzed in quadruplicates. *, p<0.05; ***, p< 0.001, as determined by two-way ANOVA for the CD8 SP +/w vs CD8 SP +/+ comparison.