Foxp3 transcription buffer set

Draining lymph nodes and tumors were collected in ice-cold PBS, and blood was collected via cardiac or tail vein puncture in heparin-containing tubes. Tissues were processed as previously described^{74 (link)}. Blood erythrocyte lysis was performed in NH₄Cl buffer for 5 min. For intracellular cytokine assessment, single cell suspensions were stimulated ex vivo with 50 ng/ml PMA, 1 μM ionomycin and Golgi-Plug (1:1000; BD) for 3 h at 37°C in IMDM medium supplemented with 8% FCS, 100 IU/ml Penicillin-Streptomycin (Roche) and 0.5% β-mercaptoethanol. For surface antigen staining, cells were incubated for 20 min with anti-CD16/32 (2.4G2, BD Biosciences), to block unspecific Fc receptor binding, and fluorochrome-conjugated antibodies diluted in FACS buffer (2.5% FBS, 2 mM EDTA in PBS). For analysis of intracellular proteins, cells were fixed and permeabilized after surface and live/dead staining using the FOXP3 Transcription buffer set (Thermofisher), according to manufacturer’s instruction. Fixation, permeabilization and intracellular staining was performed for 30 min. Data was analyzed on BD Symphony SORP or sorted on a FACS ARIA II (4 lasers). Absolute cell counts were determined using 123count eBeads (ThermoFisher) according to manufacturer’s instructions. The antibodies and viability-detection reagents used in this study are listed in supplemental table 2.

Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 10⁶ single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO₂. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).