Anti ifn γ pe clone xmg1.2

Single cell suspensions of splenocytes and lymph nodes were prepared as described above. One million cells were stimulated with PMA/Ionomycin (eBiosciences, San Diego, CA) for 5 hours at 37°C in RPMI media containing 10 % fetal calf serum, in a humidified incubator (5% CO₂, 95% air). Protein transport inhibitors (Brefeldin A+ Monesin) were added after one hour of incubation with PMA/Ionomycin (17 (link)). After 5 hours of stimulation with PMA/Ionomycin, the cells were harvested and labelled with fluorescence-conjugated antibodies to CD4 and CD8. Cells were then fixed and permeabilized with Cytofix/Cytoperm Plus (BD Biosciences, 250 μL / tube) for 20 minutes at 4°C, followed by washing with BD Perm/Wash solution. Anti-IFNγ-PE (clone XMG1.2, eBiosciences) was used to detect intracellular IFNγ production. Fluorochrome-conjugated isotype-specific IgGs served as controls. All samples were analyzed using an Accuri C6 flow cytometer (BD Biosciences, San Diego, CA). Data were analyzed using Accuri C6 software.