Additional Supporting Information may be found in the online version of this article.

22042_sm_SuppFig1.jpeg115KOnline Figure 1. Reagent Titrations. Shown are examples of two-fold serial dilutions of antibody conjugates performed on PBMC from healthy donors. For each conjugate, data from each .fcs file has been merged into one graph to facilitate comparison. Serial dilutions of 2X start at 20µL decreasing to 0.15µL for the reagents in the top two rows. In the bottom row, CD28 antibody dilutions begin at 20µL; CD4 and CD3 reagent dilutions begin at 10µL. Titers selected for use in the final panel are highlighted in fuschia.
22042_sm_SuppFig2.jpeg98KOnline Figure 2. Ranking Example. Optimal titration volumes were selected for each conjugate (outlined in fuchsia). Single-source frozen PBMC were thawed and stimulated with SEB for six hours per standard operating procedure, then stained and analyzed. Percentages shown represent the fraction of IFNγ+ CD8+ T cells at the selected titration volume. Here, representative titration graphs compare brightness, background, separation of positive and negative IFNγ+ PBMC, and the fraction of cytokine positive CD8+ T cells between various IFNγ antibody conjugates. Examples shown include monoclonal antibodies of several fluorochrome and clone options from three separate vendors. Using a scale of 1-5, where 1 represents the optimal ranking, each graph was assigned a number according to the stated criteria above. Based on brightness alone, (MFI > 10,000) Reagents 1, 2, 4, and 5 were selected for further review. Reagent 5 was eliminated due to increased background. The small degree of IFNγ separation seen with Reagent 2, as compared to the other reagents, further narrowed the selection to Reagents 1 and 4. Finally, because the highest percentage of IFNγ identified within the CD8+ T cell population was obtained with Reagent 1, which was subsequently tested in both the 7- and 10-color panels, it was selected as the conjugate of choice.
22042_sm_SuppFig3.jpeg94KOn-line Figure 3 Panel A identifies naïve and memory PBMC, which allows functional responses to be expressed as a fraction of total memory T cells. Panel B, with the inclusion of a third marker, CCR7, provides even greater differentiation of total memory into central and transitional memory populations. In panel C, CD4 and CD8 T cell memory subsets are identified by first gating on individual CD28+ and CD28- populations, and then further delineating those cell groups using CCR7 and CD45RA to distinguish naïve, central (CM) and transitional memory (TM) (CD28+) cells from effector memory (EM) and terminal effector (TE) (CD28−) PBMC.
22042_sm_SuppInfo.doc121KSupporting Information

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