Tnf α bv605

Draining s.c. LNs (axillary and popliteal) and the spleen were isolated from mice. Spleens were first mashed into a single cell suspension with plain DMEM media (Gibco 11966025), filtered through 70 μM cell strainers, and lysed with ACK lysis buffer (Gibco A1049201). LNs were digested with 1 mg/mL Ca²⁺ supplemented Collagenase D (Roche 11088866001) for 45 min at 37°C and gently mashed into a single cell suspension, also with DMEM and through 70 μM cell strainers. The cells were resuspended in IMDM media (Gibco 12440053), supplemented with 10% FBS and 1% Penicillin-Streptomycin (Gibco 15140122), and counted using a LUNA automated fluorescent cell counter (Logos biosystems). Cells were seeded at a count of 1x10⁶ - 3x10⁶ per well in 96-well round-bottom plates for subsequent antibody staining for flow cytometry. Antibodies against the following markers were used: CD3 – BUV395 (BD Biosciences 563565), CD8-BUV737 (BD Biosciences 612759), CD4 – BUV496 (BD Biosciences 612952), Foxp3 – FITC (BD Biosciences 560403), CD25 – BV605 (BioLegend 120235), ST2 – BV421 (BD Biosciences 566309), Lag3 – PerCP-Cy5.5 (BD Biosciences 564673), CTLA4 – PE-Cy7 (eBioScience 17-1522-82), IFNγ – APC (BioLegend 505810), TNFα – BV605 (BioLegend 506329), IL-2 – FITC (BioLegend 503806), IL-10 – APC-Cy7 (BioLegend 505036), PD-1 – BV711 (BioLegend 135231), Tim3 – PE (BD Biosciences 566346).

Cells isolated from spleen, lungs or BAL were cultured with 10 µg/ml Purified Protein Derivative of M. tb (PPD-T) (Statens Serum Institut, Copenhagen, DK), 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma-Aldrich) for 16 h at 37 °C/5% CO₂. Cells were washed (300 g/5 min) and surface stained for 15 min/4 °C with pre-titrated antibodies: PD-1⁻FITC, CD44-BV785, CD8-AF700, KLRG1-PerCP-Cy5.5, CXCR3-BV421, live/dead-Zombie Aqua (all BioLegend, San Diego, CA, USA), CD90.2-eFluor 450 (eBioscience), and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per the manufacturer’s instructions and stained intracellularly for 30 min/4 °C with IFN-γ-PE-Cy7, IL-2-APC (both eBioscience) and TNF-α-BV605 (BioLegend). Cells were washed again and acquired using an LSRFortessa™ analyser, utilising a 532 nm laser for PE and PE-conjugate excitation, with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star, Ashland, OR, USA) on a minimum of 100,000 live lymphocytes (50,000 for BAL).