Cd69 pe texas red

CD4⁺ and CD8⁺ T cell responses were measured from blood and tissues by flow cytometric ICS, as previously described.^{54 (link)} Briefly, 1 × 10⁶ mononuclear cells were incubated with Gag or Vif open-reading frame pools and the co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by addition of Brefeldin A (Sigma-Aldrich) for an additional 8 h. Co-stimulation without antigen served as a background control, while incubation with Staphylococcal Enterotoxin B (Toxin Technology) served as the positive control. The cells were then labeled with CD4 PE-Cy7 (Biolegend) and CD8 PerCP-Cy5.5 (BD Biosciences) and fixed with 2% paraformaldehyde. After permeabilization, the cells were stained with CD3 Pacific Blue, IFN-γ APC, TNF-α FITC (BD Biosciences, all), and CD69 PE-Texas Red (Beckman Coulter). The cells were fixed and flow cytometric analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star, Ashland, OR). In some cases, cells were CD25-depleted prior to setting up the ICS experiment to remove T regulatory cells (Miltenyi Biotec).