Cell stimulation cocktail pma and ionomycin

The polyfunctionality of CD4 T cells was assessed using intracellular cytokine staining (ICS). Brain and spleen cells were stimulated with 1X Cell Stimulation Cocktail (PMA and ionomycin) (eBioscience, USA) along with R10 media in the presence of 0.2μg CD28/49d co-stimulatory antibodies (BD) per test. Unstimulated controls were treated with volume-controlled DMSO (Sigma-Aldrich). Cells were incubated in 5% CO₂ at 37°C and after 1 hour of stimulation, protein transport inhibitors 2μl/mL GolgiPlug (Brefeldin A) and 1.3μl/mL GolgiStop (Monensin) (BD, Biosciences, USA) was added to tubes and further incubated for 3 hours at 37°C, 5% CO₂. Following stimulation, cells were stained for ICS surface markers CD3, CD4, CD8, and CD95. Subsequently, the cells were fixed using cytofix/cytoperm for 10 min DMEM supplemented with 10% HI-FBS at 4°C, then permeabilized with 1X Perm wash buffer (BD, Biosciences, USA), and stained with intracellular markers TNFα, IFNγ, and IL-2 for 45 min. Finally, cells were washed and acquired on the same day using a BD FACSymphony flow cytometer.