Transcription factor permeabilization kit

Single cell suspensions from footpads, draining lymph nodes or spleens were surface stained for phenotypic analysis. Cells were incubated for 30 minutes with appropriate amounts of antibodies in the presence of Fc receptor-blocking agent (Fc block from BD Bioscience), after which cells were washed and stained with a viability dye (eBioscience). Antibodies used were directed against mouse CD45 (GL2), CD11c (HL3), CD40 (3/23), CD3 (145-2CII), CD4 (RM4-5), CD8 (53–6.7), CD25 (PC65), CD19 (ID3), CD11b (M1/70), Ly6G (1A8-Ly6G), Ly6C (HK1.4) and NK1.1 (PK136) from BD Biosciences and eBioscience. For intracellular staining, surface-labeled cells were fixed and permeabilized with the Inside Stain Kit from Miltenyi Biotec for IFNγ labelling with an anti-IFNγ (XMG1.2) or fixed and permeabilized with the transcription factor permeabilization kit from eBioscience and stained for foxp3 (MF23 from BD Biosciences). All controls were stained with the respective isotypes. Flow cytometric data were acquired on a MACSQuant device (Miltenyi Biotec) and analysed using FlowJo software (TreeStar).

PBMCs were separated by density gradient centrifugation with Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, USA) (Fig. 1B). After lymphocyte extraction, cisplatin (5 μM) was used to treat cells in suspension, and the cells were then fixed in paraformaldehyde (PFA; final concentration 1.6%). Cells were stained with a cocktail of 30 metal isotope-conjugated antibodies against surface proteins (Table S4). After surface-protein staining, the cells were treated with the Transcription Factor Permeabilization kit (eBioscience, Santiago, USA) and then stained with anti-Tax antibody at room temperature for 30 min (Table S4). Nuclear were stained with 1 mL of 1:4000 diluted ¹⁹¹Ir/¹⁹³Ir DNA intercalator (Fluidigm Sciences, San Francisco, USA) with Maxpar Perm-S Buffer (Fluidigm Sciences, San Francisco, USA) overnight at 4 °C.