Constitutive and chemokine Vav1^−/− effector T-cell trafficking in vivo. (A) HY-specific CD4⁺ WT and Vav1^−/− T cells were labeled with PKH26 (red) or CFSE (green), respectively, and injected intravenously in syngeneic female mice. Trafficking into kidney, liver, lung, and spleen was monitored at 2, 6, and 24 hours after injection by harvesting, snap-freezing the tissue, and taking 5- to 10-μm sections. The mean number of cells from at least 6 tissue sections from at least 3 experiments was quantified by wide-field fluorescence microscopy, as described in “Wide-field fluorescence microscopy and flow cytometry.” (B,C) WT and Vav1^−/− T cells were labeled with PKH26 and injected intravenously into syngeneic female mice that had received an intraperitoneal injection of 1.2 μg CXCL10. Some mice were also injected with PBS alone (ie, no T cells) as an autofluorescence control. Mice were killed 16 hours later, and the presence of PKH26-labeled, CD4⁺ T cells was analyzed by flow cytometry. Representative dot plots are shown in panel B. The mean percentage of cells present in the peritoneal lavage (calculated by subtracting the average background migration) from the percentage of migrated cells in the presence of CXCL10 is shown in panel C. Owing to the presence of an autofluorescent population of non-T cells often detected in FL-2 (also in control mice that received saline solution), cells were double-stained with an APC-conjugated anti-CD4 antibody following harvesting, and the percentage of PKH26 (FL-2)–labeled T cells gated in the CD4⁺ T-cell population is shown in the histogram and the graph, representing cumulative data from at least 3 animals. The mean plus or minus SEM observed in samples from at least 3 animals are shown. Error bars indicate standard error (*P < .05).