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Figure S2.Comparison and validation of flow cytometry methodologies. (a) Both a goat polyclonal and a rat monoclonal antibody against Tim-3 were used in separate experiments in this study (both from R&D Systems). To confirm that the levels of expression of Tim-3 correlated, using these two antibodies, we stained PBMC from 13 HIV-1–infected individuals, comprising 7 chronic progressors and 7 acutes, separately with either monoclonal or polyclonal Tim-3. Shown is a plot of the frequency of surface Tim-3 expression on CD8+ T cells as determined by either the monoclonal antibody (x axis) or the polyclonal antibody (y axis). Consistently higher frequencies of Tim-3–expressing cells were detected using the polyclonal antibody, likely reflecting a greater sensitivity for low-level Tim-3 expression; however, the expression levels determined by both antibodies correlated very closely. (b) Throughout this study, we report CD38 expression as percentage of CD38+. Shown is a correlation between levels of CD38 expression as reported by percentage versus by MFI. (c) Throughout this study, we report Tim-3 expression as percentage of Tim-3+. Shown is the correlation between levels of Tim-3 expression as reported by percentage versus by MFI. Solid lines show the mean.