Pe anti mouse cd223 lag 3 antibody

PerCP-Cy5.5 conjugated antibody to CD3 (cat 45–0031-82, clone 145-2C11); APC or APC-eFluor780-anti-CD8 (cat 17–0081-82, 47–0081-82, clone 53–6.7); eFluor450-anti-CD279 (PD-1) antibody (cat 48–9985-82, clone J43), PE-Cy7 anti-IFNγ (cat 25–7311-82, clone XMG1.2); PE-anti-Ki-67 (cat 12–5698-82, clone SolA15), APC-eFluor780 labelled antibody to CD44 (cat 47–0441-82, clone IM7); and PE-Tim3 (cat 12–5870-82, clone RMT3-23) were obtained from eBioscience. PE-anti-mouse CD223 (LAG-3) antibody (cat 125,208 clone C9B7W), APC Annexin V (cat 640,920), and Propidium iodide (PI, cat 421,301) were from Biolegend. For analysis of IFNγ producing cells, the cells were stimulated with 50 ng/ml PMA and 200 ng/ml ionomycin in the presence of Golgistop (BD Biosciences) for 3 h and then fixed with the Foxp3 staining kit (eBioscience) and analysed by flow cytometry. For all flow cytometry analyses, data were acquired on a LSR II or Canto II (BD Immunocytometry Systems) and analysed using FlowJo (Tree Star).

To isolated lung lymphocytes, mice lungs were collected in complete culture RPMI-1640 medium. Lung tissues were minced gently in complete medium, and the filtrates containing lung lymphocytes and other inflammatory cells were collected. The viability of the freshly isolated cells was evaluated by the Trypan blue dye exclusion test. PBMCs were isolated from blood samples using a Ficoll gradient (Axis Shield, Norway). PBMCs or lung cells were stained with APC anti-mouse CD19 antibody (BioLegend cat # 115512), APC/Cyanine7 anti-mouse CD138 (Syndecan-1) antibody (BioLegend cat # 142530), PerCP/Cyanine5.5 anti-mouse CD1d antibody (BioLegend cat # 123514), APC anti-mouse CD25 antibody (BioLegend cat # 102012), and PE anti-mouse CD223 (LAG-3) antibody (BioLegend cat # 125208). After surface staining, cells were stained with PE anti-mouse IL-27 p28 antibody (BioLegend cat # 516908), PE/Cy7 anti-mouse IL-10 antibody (BioLegend cat # 505026), and FITC anti-mouse FOXP3 antibody. Corresponding isotype controls were used for each antibody. All cells were analyzed with BD LSR II flow cytometer using DIVA software version 8.0. The corresponding data is deposited in S file.