CXCR4 signaling is the main pathway involved in the chemotactic migration of B-ALL cells induced by a TCM. (A) Workflow. After removal of the tunica albuginea, pieces of testis tissue were incubated in RPMI 1640 containing 1% bovine serum albumin for 90 minutes at 37°C. The TCM was collected by centrifugation and still contained mouse testis cells. The bottom left panel illustrates the CD10/CD19-negative status of testis cells still present in TCM obtained by flow cytometry. REH cells that had been incubated with plerixafor (20 μM), NSC23766 (25 μM), anti-CD9 blocking antibody, or IgG isotype control (1 μg/mL), were allowed to migrate in a Boyden chamber in response to TCM. After 5 hours, the cells present in the lower chamber of the migration system were labeled with human anti-CD10 and anti-CD19 antibodies and subjected to flow cytometry. The migration rate was defined as the percentage of human cells detected in the lower chamber after migration. The bottom right panel illustrates the results obtained for a set of experiments with REH cells. (B) The migration rates of 3 independent experiments (•, ♦, ∆) are presented using a scatter dot plot representation with dotted lines connecting the results obtained in the same set of experiments. The histograms indicate the mean for each condition. (C) REH CD9^high, CD9^medium, and CD9^low cells migration in response to TCM was tested. The results are presented using a scatter dot plot where the histograms represent the means of 6 independent experiments. *P < .05 in Wilcoxon test. (D) NALM6 cells migration in response to TCM after a pretreatment with plerixafor, NSC23766, and anti-CD9 antibody was tested. The scatter dot plot represents the results of 3 independent experiments (•, ♦, ∆) and the dotted lines connect the results obtained in the same set of experiments. The histograms indicate the mean for each condition.