Anti mouse ifn γ xmg1.2

Lung single-cell suspensions from vaccinated or Mtb-infected mice were generated as before4 (link). For LNs and spleens, organs were passed through a 70 μm cell strainer and then processed as for lungs4 (link). For flow cytometric analysis, cells were either stained immediately, or stimulated for 18 h in the presence of Ag85B_240–254 peptide, with GolgiStop (5 μl ml⁻¹; BD Biosciences) and Brefeldin A (1 μg ml⁻¹; BioLegend, San Diego, CA, USA) added for the last 5 h. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were collected using a BD FACS Jazz or a BD LSR Fortessa, and analysis was performed using FlowJo (Treestar, Ashland, OR, USA). Antibodies used include anti-mouse CD11c (clone HL3; BD Biosciences; dilution: 1/200), anti-mouse MHC-II (clone M5/114.15.2; Tonbo Biosciences, San Diego, CA, USA; dilution: 1/150), anti-mouse CD3 (clone 500A2; BD Biosciences; dilution: 1/200), anti-mouse CD4 (clone RM4-5; BD Biosciences, dilution: 1/200), anti-mouse CD44 (clone IM7; eBioscience, dilution: 1/400), anti-mouse IL-17 (clone TC11-18H10; BD Biosciences, dilution: 1/100), anti-mouse IFN-γ (XMG1.2; BD Biosciences; dilution: 1/100), anti-mouse Thy1.1 (clone OX-7; BD Biosciences; dilution: 1/100) and anti-mouse CD103 (clone 2E7, eBioscience; dilution: 1/200).

Cells were stained with the indicated antibodies for 30 min at 4°C. The following antibodies for flow cytometry were obtained from BioLegend, CA, United States: anti-mouse CD4 (GK1.5), anti-mouse CCR6 (29-2L17), anti-mouse CCR9 (9B1), anti-mouse CD36 (HM36), and anti-human CD4 (OKT4). For intracellular cytokine analysis, cells were stimulated with PMA and ionomycin for 30 min and treated with brefeldin A for 4 h before staining Fixation/Permeabilization Solution Kit (BD, NJ, United States) followed by anti-mouse TNF-α (MP6-XT22, BioLegend, CA, United States), anti-mouse IFN-γ (XMG1.2, BD, NJ, United States), anti-human TNF-α (Mab11, BioLegend, CA, United States), or anti-human IFN-γ (4S.B3, BioLegend, CA, United States). Cell apoptosis was detected with an Annexin V staining kit (BD, NJ, United States) according to the manufacturer’s instructions. For cell proliferation, cells were labeled with 5 μM CFDA-SE (CFSE) (Invitrogen, CA, United States). Seventy-two hours later, they were collected and analyzed by flow cytometry. Acquisition was carried out on FACSVerse (BD, NJ, United States), and analysis was performed with FlowJo v10 software.