Fitc labeled anti cd4

Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described (Zhao et al., 2013 (link); Cossarizza et al., 2017 (link); Dong et al., 2018 (link)). Briefly, the excised right lung was digested by using lung dissociation kits (MiltenyiBiotec Technology & Trading Co. Ltd, Shanghai, China) to prepare single cell suspensions following the instructions. And then the cells were stained with the monoclonal anti-murine fluorochrome-conjugated Abs and detected by an Attune NxT instrument (Life Technology). Besides, the antibodies used were bought from BioLegend, including eflour506-labeled anti-CD45, Zombie NIR™ Fixable Viability Kit, Brilliant Violet 421™-labeled anti-CD11b, APC-labeled anti-Siglec-F, PE-labeled anti-Ly-6C, APC-labeled anti-CD25, FITC-labeled anti-CD4, PE-labeled anti-IL-17. PE-labeled anti-Foxp3 was purchased from eBioscience.

FITC-labeled anti-CD4 (clone: GK1.5), FITC-labeled anti-CD19 (clone: 6D5), PE-labeled anti-B220 (clone: RA3-6B2), PE-labeled anti-CD95 (clone: SA367H8), PE-labeled anti-CD44 (clone: IM7), PE/Cy7-labeled anti-IgD (clone:11-26c.2a), PE/Cy5-labeled anti-CD62L (clone: MEL-14), APC-labeled anti-IgG (clone: Poly4053), APC-labeled anti-CD138 (clone: Syndecan-1), APC-labeled anti-CD80 (clone: 16-10A1), and Pacific Blue-labeled anti-GL7 (clone: GL7) were purchased from BioLegend. CD16/CD32 purified antibody (clone: 93), and FITC-labeled anti-CD38 (clone: 90) were purchased from eBioscience.

The PE-labeled MHC-II Ag85B tetramer I-A(b)/FQDAYNAAGGHNAVF and control tetramer I-A(b)/PVSKMRMATPLLMQA were provided by the NIH Tetramer core facility at Emory University (Atlanta, GA). Isolated PBMCs, splenocytes, and lung lymphocytes were incubated with fixable viability stain 780 (BD Biosciences), APC-labeled anti-CD3 (Biolegend), PerCP-Cy5.5-labeled anti-CD8 (eBioscience), FITC-labeled anti-CD4 (Biolegend), and BV510-labeled anti-CD44 (Biolegend), together with either control or Ag85B PE-labeled MHC-II tetramer. To detect tissue-resident CD4 T cells in the lung, lung lymphocytes were incubated with the same cocktails, together with APC-Cy7-labeled anti-CD69 (Biolegend). Cells were washed three times with PBS/2% PBS. Sample acquisition was performed on FACSCelesta and data analyzed using FlowJo.