Apc anti mouse cd25 antibody

Mouse blood cells were harvested and divided into three parts. First, spleen cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD3 antibody (100306, BioLegend, San Diego, USA), 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (100407, BioLegend), and 100 μL of 12.5 μg/mL APC anti-mouse CD8 antibody (100732, BioLegend) at 25°C for 30 min, washed, then incubated with 100 μL of 10 μg/mL FITC goat anti-mouse IgG antibody (405305, BioLegend) at 25°C for 30 min in the dark. Second, blood cells were treated with 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (BioLegend), 100 μL of 25 μg/mL APC anti-mouse CD25 antibody (101910, BioLegend), and 100 μL of 12.5 μg/mL PE anti-mouse Foxp3 antibody (320008, BioLegend) via the same protocol described above. Third, blood cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD19 antibody (115506, BioLegend) using the same protocol described above. After incubation, cells were washed and resuspended in 0.5 mL of PBS/2% paraformaldehyde and quantified by flow cytometry (BD Calibur^TM).

CSPCM was provided by HeBei TaiFeng Biotechnology Co., Ltd. (Handan, China). CTX CAS#6055-19-2(C₇H₁₅Cl₂N₂O₂P·H₂O) was purchased from Source Leaf Biotechnology Co., LTD(Shanghai, China). Eastep® Super Total RNA Extraction kit was purchased from Promega (Beijing) Biotechnology Co., LTD (Beijing, China). PrimeScript™ RT reagent Kit with gDNA Easer, TB Green® Premix Ex Taq™ II(Tli RNaseH Plus) were purchased from Takara Biomedical Technology (Beijing) Co., LTD (Beijing, China). BCA Protein Assay Kit was purchased from Beijing ComWin Biotechnology Co., LTD. (Beijing, China). SDS-PAGE Gel preparation kit was purchased from Biomed Genentech Inc. (Beijing, China). Rabbit Anti-Foxp3 Polyclonal Antibody, Rabbit Anti-RORγt Polyclonal Antibody and Mice Anti-GAPDH Polyclonal Antibody were purchased from Beijing Bioss Biotechnology Co. LTD (Beijing, China). FITC anti-mouse CD3 Antibody, APC anti-mouse CD4 Antibody, PE anti-mouse IL-17A Antibody, FITC anti-mouse CD4 Antibody, APC anti-mouse CD25 Antibody, PE anti-mouse FOXP3 Antibody, True-Nuclear™ Transcription Factor Buffer Set, True Nuclear TM 4X Fix Concentrate, True Nuclear TM 10X Per and True Nuclear TM Fix Diluent were purchased from BioLegend, Inc (California, United States).

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.

AT-infiltrating stromal cells were stained with FITC anti-mouse CD3ε antibody, PE anti-mouse CD4 antibody, PE/Cy7 anti-mouse CD25 antibody, APC anti-mouse CD8a antibody, FITC anti-mouse CD127 antibody, PerCP/Cy5.5 anti-mouse CD4 antibody, APC anti-mouse CD25 antibody (these antibodies were purchased from Biolegend, San Diego, CA, USA), PE anti-mouse/Foxp3 antibody (Invitrogen, Carlsbad, CA, USA), PE anti-mouse CD11b antibody (Biolegend), PE/Cy7 anti-mouse Ly-6C antibody (Biolegend, San Diego, CA, USA) and APC anti-toll-like receptor 4 (TLR4) antibody (Invitrogen, Waltham, MA, USA), and analyzed using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).