FIGURE 3.

Determination of total versus cell surface-bound CD226 protein and CD226-specific mRNA. A, real-time PCR analysis of CD226-specific mRNA present in SP thymocytes and naïve peripheral T cells. Cells were sorted by flow cytometry and the CD226 mRNA quantified as described under “Experimental Procedures.” Each dot represents cells isolated from one animal. n.s., not significant. B, peripheral CD4⁺ T cells or CD8⁺ T cells purified by magnetic bead separation as described under “Experimental Procedures.” Cells were then either stained directly for detection of CD226 (surface staining) or fixed and permeabilized prior to CD226 staining (total CD226 levels; permeabilized cells). Shown are mean fluorescence intensities (MFI) because staining with isotype control mAb caused a uniform but elevated level of background when applied on fixed/permeabilized cells. Each dot represents cells isolated from one animal. The summarized result from two experiments is shown. The mean fluorescence elicited by staining of CD4⁺ T cells was set at 100 in each experiment. WT, +/+; CD155^−/−, −/−; ***, p < 0.001.