Fix perm intracellular staining kit

Splenocytes were restimulated ex vivo in complete media with either 30nM p79 MHV-specific peptide or with 1 μg/ml PMA and 1 μg/ml Ionomycin (Sigma Life Sciences) in the presence of Brefeldin A (BD Biosciences) at 37°C for 4h. Subsequently, cells were surface stained followed by fixation with the Fix/Perm intracellular staining kit (BD Pharmingen) at 4°C for 20 min, and then stained with antibodies against IFN-γ (PE-Dazzle, BioLegend, clone XMG1.2), and TNF (APC, Biolegend, clone MP6-XT22) per manufacturer’s instructions. For assessment of degranulation activity, an anti-CD107a antibody (BV421, Biolegend, clone 1D4B) was added along with the stimuli outlined above, in the presence of Brefeldin A and Monensin (BD Biosciences) and cultured at 37°C for 4h followed by surface staining at 4°C for 20 min.

Cells isolated from spleens and graft-draining axillary and brachial lymph nodes (dLN) were stained with anti-CD4 (BD Biosciences), anti-CD8 (Invitrogen) and anti-Thy1.1 (BD Biosciences). For phenotypic analysis cells were also surface-stained with anti-PD-1 (BioLegend), anti-2B4 (BD Biosciences or eBioSciences), anti-Thy1.1 (BD Biosciences), anti-LAG-3 (BioLegend), anti-CD127 (BioLegend), anti-KLRG-1 (eBioSciences), anti-CD44 (BioLegend or BD Biosciences), and anti-CD48 (BioLegend). Absolute numbers of lymphocytes from the spleen and draining lymph nodes were calculated using a Cellometer Auto T4 Cell Viability Counter (Nexcelom) according to the manufacturer’s instructions. Samples were analyzed on an LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.). For intracellular cytokine staining, lymphocytes were restimulated ex vivo with 1 μg/mL phorbol 12-myristate 13-acetate (PMA) (Sigma Life Sciences) and 1 μg/mL ionomycin (Sigma Life Sciences) where indicated, in the presence of 1 μg/mL Brefeldin A (BD Biosciences) for 4 hours. The Fix/Perm intracellular staining kit (BD Pharmingen) was used to detect IL-2 (BD Biosciences), TNF (BioLegend), and IFN-γ (BD Biosciences), according to manufacturer’s instructions.

Cells were cultured for 4 hours in 96-well round-bottomed plates at a concentration of 1-2.5 × 10⁶ cells/well in 0.2 mL of complete medium and restimulated ex vivo with 1 μg/mL PMA (Sigma Life Sciences) and 1 μg/mL Ionomycin (Sigma Life Sciences) where indicated, in the presence of 1 μg/mL Brefeldin A (BD Biosciences) for 4 hours. The Fix/Perm intracellular staining kit (BD Pharmingen) was used to detect IL-2 (BD Biosciences), TNF (BioLegend), and IFN-γ (BD Biosciences), according to manufacturer’s instructions.