Flow cytometry gating method used in the analysis of CD8+ T-cell responses.

<p>Shown is a typical rectal mucosal cell preparation that was stimulated with HIVgag peptides. After initial estimation of a lymphocyte gate based on forward versus side scatter, viable cells (7-AAD negative) were carried forward for selection of CD3+CD8+ cells. The resulting CD3+CD8+ population was further gated based on positivity for each of the 5 functional responses measured: MIP-1β, TNF-α, IFN-γ, IL-2 and CD107a. The frequencies of cells in these populations were corrected using background values (costimulation only) and then compiled and analyzed directly. For more detailed analyses, the 5 populations were instead entered into Boolean gating analysis to generate 31 populations consisting of all possible combinations of functionality (the 32^nd population representing cells negative for all 5 functions was ignored). Background correction was applied after Boolean gating (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003577#s4" target="_blank">Methods</a>).