The inhibitory control of CCR2 on migration is independent of the IFN-γ regulated pathway. (A) IFN-γ transcription in CCR2^–/– cells. Mature (IgD⁺ B220⁺) and immature (IgD^– B220⁺) B cells derived from control or CCR2^–/– mice were purified, total RNA was isolated, and reverse transcription was carried out by using Superscript II RT. (B-C) Actin polymerization assay. Mature B cells were treated for 30 minutes with medium or supernatant collected from immature B cells of control or CCR2^–/– mice (B). Immature CCR2^–/– B cells were pretreated with IFN-γ (0.1 U/mL) for 30 minutes (C). The cells were then stimulated with 50 μg/mL SDF-1 for 15 seconds, fixed, and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. Percentage increase in actin polymerization was calculated as described in Figure 2. (D) Transwell migration assay. Immature CCR2^–/– B cells were pretreated with IFN-γ (0.1 U/mL) for 30 minutes and then placed in the upper well of a 24-well transwell plate in the presence or absence of SDF-1. After 3 hours, the number of the migrating cells found in the lower chamber was evaluated by FACS analysis. Percentage migration was calculated as described in Figure 2. The results presented are representative of 3 different experiments. Error bars represent the standard error of the results of all the experiments used to calculate the average. *Significant at a 98% level.