Pe anti ifn γ clone xmg1.2

Presence of activated antigen-specific CD8⁺ and CD4⁺ T cells after immunization was determined by flow cytometric analysis of cells after intracellular cytokine staining assay. Cell were stimulated overnight with gp140 protein and then incubated further with GolgiPlug reagent (BD Biosciences, San Jose, CA) for 6 hours prior to cellular staining. Cells were first stained for surface markers and then permeablized for staining with PE-conjugated IFN-γ antibody (BD Biosciences, San Jose, CA) in 1× Perm/Wash Buffer (BD Biosciences, San Jose, CA)(17). Samples were run on the LSRII flow cytometer and analyses were performed using FlowJo software (Tree Star Inc, Ashland, OR). The monoclonal antibodies used were PB-anti-CD3 (clone 500A2), PerCPCy5.5-anti-CD8 (clone 53-6.7) APC-anti-CD4 (clone RM4-5) and PE-anti-IFN-γ (clone XMG1.2), all purchased from BD Biosciences, San Jose, CA. For exclusion of cells with background fluorescence for PE, an aliquot of cells used for fluorescence-minus-one (FMO) from each tissue was stained with anti-CD3, anti-CD8 and anti-CD4 antibodies and used as control for establishing gates for CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ cell population. Percentage of antigen specific activated cells within CD4⁺ and CD8⁺ lymphocyte subsets was determined for animals receiving immunization with either gp140 protein alone or gp140 + adjuvants.