Fixable viability stain 510 antibody

GREER laboratories supplied HDM; OVA, LPS, and aluminum hydroxide gel from Sigma; mice spirometer (MAX 1320) from Buxco®, USA; Beijing Zhongshidichuang Science and Technology Development Co. Ltd. supplied animal asthma inducing instrument (YLS-8A); centrifuge (5810 R) from Eppendorf®, Germany; image analyzer (Leica Application Suite V4) from Leica, Germany; and Olympus, Japan (Leica®) supplied optical microscope (DMI3000B); 150-mesh cell sieve (Biosharp, BS-100-XBS, China); bronchial epithelial growth medium (Procell, CM-M007, China); DAPI and cytokeratin specific monoclonal antibody (pan-Cytokeratin, Santa Cruz, sc-8018, USA); leukocyte activation cocktail (550583, BD Biosciences, USA); cell viability marker (Fixable Viability Stain 510 antibody, BD Pharmingen); PE-anti-IL-4 (BD Pharmingen); APC-anti-IL-17A (Biolegend); Lipofectamine 3000 (Invitrogen, USA); bicinchoninic acid (BCA), Beyotime, Shanghai, China; DHT (B8214) and 17β-estradiol (C4348) APExBIO, USA; MBD2 antibody (Abcam, Cambridge, USA); eosinophil antibody (anti-ECP, Biorbyt, Cambridge, UK); neutrophil antibody (anti-Gr-1, Biolegend, San Diego, USA); GATA3 (10417-1-AP) and β actin (60008-1-Ig) Proteintech, USA; RORγt (EPR20006) abcam, USA.

CD4⁺ T cells were stimulated with 2 μL/ml (1 × 10⁶ cells/ml) leukocyte activation cocktail (550583, BD Biosciences, United States ), and cultured at 37°C with 5% CO₂ for 6 h, then collected for flow cytometry analysis. After a 6-h incubation, cells were stained with a marker of cell viability (Fixable Viability Stain 510 antibody, BD Pharmingen) for 15 min at room temperature in the dark. Then, cells were stained for surface markers with FITC-anti-CD4 antibody (Biolegend) followed by fixation and permeabilization using the Cytofix/Cytoperm Soln Kit (BD Pharmingen) for 30 min at 4°C in the dark. After washing with permeabilization buffer, cells were stained for intracellular markers with APC-anti-IL-17 A (Biolegend) and PE-anti-IL-4 (BD Pharmingen) antibodies in permeabilization buffer for 30 min at 4°C in the dark. Isotype controls were employed in the control group. Flow cytometry was performed, and data were analyzed using FACS CantoⅡ(Becton Dickinson) and FlowJo version X software.

CD4+ T cells were stimulated with 2µl/ml (about 1 × 10⁶ cells /ml) of leukocyte activation cocktail (BD Biosciences) and collected after cultured at 37°C and 5% CO2 for 4-6h. First, cells were stained at room temperature for 15 minutes in the dark with a cell viability marker (Fixable Viability Stain 510 antibody, BD Pharmingen). Second, cell surface markers were stained with FITC - anti-CD4 (Biolegend) antibody, followed by fixation and permeability at 4°C for 30 min in the dark using Cytofix/Cytoperm Soln Kit (BD Pharmingen). Finally, APC-anti-IFN-γ (Biolegend) and PE-anti-IL-4 (BD Pharmingen) antibodies were stained in osmotic buffer at 4°C for 30 min in the dark to detect intracellular markers. The normal control group was homologous. Then, flow cytometry was conducted, and the data were analyzed using the FACS Canto Ⅱ (Becton Dickinson) and FlowJo V10 software.