Pe labeled anti il 12p35 or control murine igg1 mab

Peritoneal exudate cells or cultured cells were washed in FACS buffer (plus 2% FBS), followed by incubation with anti-CD16/CD32 (eBioscience, San Diego, CA, USA) for Fc blocking. We stained cells with anti-CD11b, anti-F4/80, and anti-Ly6C (HK1.4) labeled with PE, FITC or allophycocyanin (BD Biosciences, Chicago, IL, USA), or with Alexa Fluor 488-labeled anti-CD301 (MGL) mAb (AbD Serotec, Kidlington, UK), or control rat IgG2a mAb (R&D Systems, Minneapolis, MN, USA). For intracellular staining, we washed, permeabilized, and stained cells with PE-labeled anti-IL-12p35 or control murine IgG1 mAb (R&D Systems), anti-NOS2 or control rat IgG2a mAb (BD Bioscience), or with FITC-labeled anti-arginase 1 or control sheep IgG mAb. Cells were washed, fixed, and acquired with the CellQuest software, on a FACSCalibur system (BD Biosciences). For analysis, we used the FlowJo software (TreeStar, Ashland, OR, USA). For IL-12p35⁺, NOS2⁺ (M1), and CD301⁺, arginase⁺ (M2) subsets, gates were based on the exclusion of background staining with isotype control mAbs, as in Figure 2B. For evaluation of Lm-RFP infection, PECs or cultured macrophages were first stained with allophycocyanin-anti-F4/80 and then analyzed for Lm-RFP⁺ cells within gated F4/80⁺ macrophages.

PECs or cultured cells were washed in FACS buffer (plus 2% FCS), followed by incubation with anti-CD16/CD32 (eBioscience, San Diego, CA, USA) for Fc blocking. We stained cells with anti-CD8, anti-CD5, anti-CD19, anti-B220, anti-F4/80 mAbs, anti-CD11b, and anti-CD11c (clone HL3) labeled with PE, FITC, or allophycocyanin (BD Biosciences, Chicago, IL, USA), or with FITC-labeled anti-Ly6G (clone 1A8), allophycocyanin-labeled anti-Ly6C (clone HK1.4), or anti-CD4 mAbs (eBioscience), or with Alexa Fluor 488-labeled anti-MGL1 (CD301a) mAb (AbD Serotec, Kidlington, UK) or control rat IgG2a mAb (R&D Systems, Minneapolis, MN, USA). For intracellular staining, we washed, permeabilized, and stained cells with PE-labeled anti-IL-12p35 or control murine IgG1 mAb (R&D Systems). For IL-12p35 (M1) and MGL1 (M2) subsets, gates were based on the exclusion of background staining with isotype control mAbs. We then washed, fixed, and acquired cells with the CellQuest software on a FACSCalibur system (BD Biosciences). For apoptosis detection, cells were first stained with allophycocyanin anti-CD8, washed, and then treated with FITC-annexin V (BD Biosciences) for 20 min at room temperature, according to the manufacturer; 7-AAD (eBioscience) was added just before flow cytometry. For analysis, we used the FlowJow software (TreeStar, Ashland, OR, USA).