CCR2-deficient immature B cells exhibit elevated actin polymerization and chemokine-induced migration. (A) Splenocytes from control and CCR2^–/– mice were triple stained with anti-B220 and anti-IgD or anti-IgM, anti-CD23, anti-CD21, and anti-CD5. Dot plots show the expression of the markers on B220⁺ cells. M indicates mature B cells; T1, transitional B cells 1, T2, transitional B cells 2; MZ, marginal zone B cells. Histograms show the expression of CD5 on B220 cells. Numbers represent percentage of each population. The results presented are representative of 5 different experiments. (B) Purification of immature B cells. IgD^– splenocytes from control and CCR2^–/– mice were separated by anti-B220 magnetic beads as described in “Materials and methods.” Numbers represent percentage of CD5-positive cells. (C) Transwell migration assay. Immature (IgD^– B220⁺) B cells from control or CCR2^–/– mice were placed in the upper well of a 24-well transwell plate in the presence or absence of SDF-1 (1000 ng/mL). The number of the migrating cells found in the lower chamber was evaluated after 3 hours of FACS analysis. Percentage migration was calculated as the number of migrating cells in the lower chamber as a fraction of the input cells in the upper chamber. The results presented are representative of 3 different experiments. (D) Cytoskeleton rearrangement. Immature B cells from control and CCR2^–/– mice were stimulated with 500 ng/mL SDF-1 for 15 seconds, fixed, and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. The change in polymerized actin was analyzed by FACS. Percentage increase in actin polymerization was calculated as the polymerization of actin in the presence of SDF-1–polymerization of actin without SDF-1/polymerization of actin without SDF-1. The results presented are representative of 5 different experiments. Error bars represent standard error of the results of all the experiments used to calculate the average.