Permeabilization solution

Single cell suspensions were obtained from spleens of recipient mice. Cells were labeled for surface and intracellular antigens with fluorescence-labeled anti-mouse antibodies (eBioscience, CA). Intracellular staining for Foxp3 was performed with a commercially available staining kit including permeabilization solution and buffer (eBioscience, CA). For intracellular cytokine staining, total splenocytes were seeded on 96-well plates and stimulated in complete media for 4 hrs at 37°C with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich), Ionomycin (500ng/ml; Sigma-Aldrich) and Brefeldin A (eBioscience). Subsequently, cells were fixed, permeabilized (BD Biosciences, CA) and stained with respective antibodies. Flow cytometry measurements were performed on a FACS Canto II (BD Bioscience, CA) and data were analyzed using FlowJo (FlowJo Software, OR).

Cryopreserved BAL cells were thawed and incubated in RPMI 1640 media containing 10% FBS at 37°C for overnight resting. Cells were incubated in the absence or presence of PMA (20ng/ml) and ionomycin (1 ug/ml) for 4 hours. A protein transport inhibitor-Brefeldin A (BFA, 5ug/ml) was added to the culture of unstimulated (treated with BFA alone) and stimulated (treated with BFA, PMA and ionomycin) samples for 4 hours. Cells were harvested and stained for viability with 5uM cisplatin (Fluidigm) for 5 minutes at room temperature before quenching with CyTOF Buffer (Fluidigm). Cells were then incubated with Fc Receptor Blocking solution (Human TruStain FcX, Biolegend) for 10 min at room temperature and stained with cell surface antibody cocktail for 45 minutes on ice. Samples were washed with CyTOF Buffer and fixed at 4°C overnight with 2% paraformaldehyde. On the following day, cells were permeabilized (permeabilization solution from eBiosciences) and stained with antibodies for 6 cytokines for 45 minutes at room temperature. Cells were washed and incubated in 0.25μM Ir intercalator (Fluidigm) for 20 min at room temperature, and stored in PBS at 4°C until acquisition. Antibodies were purchased from DVS Biosciences or conjugated in-house using purified antibodies and Fluidigm conjugation kit following manufacture’s instruction. All the antibodies were titrated prior to use.

Cell pellets were washed with phosphate-buffered saline and fixed in fixation buffer (Biolegend, San Diego, CA, USA), permeabilized with permeabilization solution (eBioscience), and then incubated with mouse LMP1 CS1-4 and rabbit p-IRF4(Y121/124) antibodies for 40 min. After washing with flow cytometry buffer, cells were incubated further with anti-mouse IgG APC and anti-rabbit IgG PE for 25 min, and then followed by flow cytometry analysis with a Flow Cytometer (Accuri, BD Biosciences, Franklin Lakes, NJ, USA) and BD Accuri C6 Software (BD Biosciences, San Jose, CA, USA). Data were further analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). The fluorescence minus one strategy was used to determine background level and to adjust multicolor compensation for cell gating.