A B C D CD4+ CD8+ Control Isotype Ab Anti-TNFa Ab Control Isotype Ab

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A B C D CD4+ CD8+ Control Isotype Ab Anti-TNFa Ab Control Isotype Ab Anti-TNFa Ab E F G H Supplementary Figure 2. Flow cytometric gating strategies for intracellular cytokine analysis. Lung cells isolated at day 10 post infection were stimulated for five hours in vitro with PMA and Ionomycin prior to staining for flow cytometric analysis. Lmphocytes were gated (A) and the doublets removed using a pulse width gate (B). Dead cells were removed using a Live/Dead gate (C) before gating the CD4+ and CD8+ lymphocyte populations (D). Cytokine expression by both CD4+ (E & F) and CD8+ (G & H) T cells was determined by the use of isotype control antibodies. IFNg secretion by CD4+ and CD8+ T cells was also determined using this gating strategy.