Flow cytometry

To track Il10 (Thy1.1) expression in the lungs of 10BiT IL-10 reporter mice during M. tuberculosis infection, we prepared single-cell homogenates, as described previously (29), washed in PBS (Life Technologies) and stained according to manufacturer’s instructions to exclude dead cells using a Live/Dead fixable red dead cell stain kit (Invitrogen). Cells were pretreated for 10 min with anti-FcgRI/FcgRII (anti-CD16/CD32) Ab. Cells were then stained with anti-Thy1.1 (HIS51; eBioscience) and other Abs against the following extracellular markers to identify myeloid cells and lymphocytes, as described previously (50, 51). Myeloid cell markers included Ly6G (1A8; BD), Ly6C (HK1.4; eBioscience), Thy1.2 (53-2.1; eBioscience), CD11c (HL3; BD), CD11b (M1/70; BD), F4/80 (BM8; eBioscience), and MHC class II (M5/114.15.2; eBioscience). Lymphoid cell markers included Thy1.2 (53-2.1; eBioscience), CD3 (145-2C11; eBioscience), CD4 (RM4-5; eBioscience/BD), CD8 (53-6.7; eBioscience), γδ TCR (GL3; eBioscience), and CD19 (eBio1D3 [eBioscience]; 6D5 [BioLegend]). In some experiments, anti-CD44 (1M7; eBioscience) Ab was also used. For intranuclear transcription factor expression, cells were stained with anti-Foxp3 (FJK-16s; eBioscience) and anti-Tbet (4B10; BioLegend) Abs using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Isotype control eBR2a (eBioscience) and MOPC-21 (BioLegend) were used as negative control. For cytokine analysis, cells were restimulated ex vivo with M. tuberculosis tuberculin purified protein derivative (PPD; 20 μg/ml; Statens Serum Institute) and anti-CD28 (2 μg/ml, clone 37.51; Harlan) for 20 h. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added during the last 4 h. After extracellular staining, cells were fixed and treated with permeabilization buffer (BD) according to manufacturer’s instructions and stained with anti–IFN-γ (XMG1.2; eBioscience) or isotype control (eBRG1; eBioscience) Abs. All stained samples were fixed with stabilizing fixative (BD) and refrigerated in the dark overnight before being acquired on a CyAN ADP analyzer (Dako, Ely, U.K.) using Summit software (Cytomation). Data were analyzed using FlowJo software (Tree Star).