Percp cy5.5 conjugated anti ifn γ

Suspensions of 1 × 10⁶ splenocytes were seeded in 100 μl RPMI medium in 96-well round bottom plates and infected by wt or receptor mutants of DK/96 or SC/18 virus at a multiplicity of infection (MOI) of 0.1, as described (Jeisy-Scott et al., 2012 (link)). After 1 h incubation with the virus, 50 μl RPMI containing 40% FBS and 400 U/ml Penicillin and 400 μg/ml streptomycin solution was added. Four days post-infection, the cells were collected and re-stimulated with plate-bound anti-CD3 (10 μg/ml; clone 145-2C11; eBioscience, San Diego, CA), and anti-CD28 (2 μg/ml, clone 37.51; eBioscience) for 6 h in the presence of BD GolgiStop™ and GolgiPlug™ protein transport inhibitors (BD Bioscience, San Jose, CA) to enhance intracellular cytokine staining. Cells were surface stained with PE-Cy™7-conjugated anti-CD4 (clone GK1.5; eBioscience) and Alexa Fluor 700-conjugated anti-CD8 (53-6.7; BD Bioscience) for 20 min at 4 °C. Cells were then made permeable with Cytofix/Cytoperm (BD Bioscience) followed by intracellular staining with PerCP-Cy5.5-conjugated anti-IFN-γ (BioLegend, San Diego, CA) for 45 min at 4 °C. Cells were washed twice with perm/wash and re-suspended in PBS/10% FBS. Samples were analyzed using an LSR II flow cytometer (BD Biosciences, Sam Jose, CA), and the cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR).