Pe cy7 rat anti mouse il 4

Splenic lymphocytes were isolated and stimulated with 2 μg/mL of the spike protein peptide pool and brefeldin A (1:1,000 dilution, Biolegend, USA) at 37°C and 5% CO₂ for 6 h. After stimulation, the splenocytes were rinsed and stained with fixable viability stain and 780 the following antibodies: FITC rat anti-mouse CD8a antibody, BV510 rat anti-mouse CD4 antibody and BV421 hamster anti-mouse CD3e antibody (BD Biosciences, USA). The cells were rinsed twice with 1× PBS, fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences). After rinsing with Perm/Wash buffer (BD Biosciences), the cells were stained with BB700 rat anti-mouse tumour necrosis factor (TNF), APC rat anti-mouse IL-10, PE-Cy7 rat anti-mouse IL-4, BV605 rat anti-mouse interleukin (IL)-2 and PE-conjugated rat anti-mouse IFN-γ (BD Biosciences). The cells were successively rinsed with Perm/Wash buffer, resuspended in 1× PBS, and determined using a FACS Lyric flow cytometric analyser (BD Biosciences). For each sample, 200,000 events were recorded, and data analysis was performed with FlowJo software (TreeStar, USA). CD4⁺ and CD8⁺ T cells were obtained by gating single cells (FSC-A versus FSC-H), lymphocytes (FSC-A versus SSC-A), and live CD3⁺ T cells (CD3⁺ versus LD780⁻). All results are expressed as the percentage of cytokine+ cells in CD4⁺ or CD8⁺ T cells.

To assess the recruitment of IL-4⁺ CD4⁺ T cells, live cells were isolated from splenocytes and LLN from allergic airway inflammatory mice that were or were not sensitized to the A. pegreffii crude extract. The cell preparation method was the same as that described in Section 2.8. Samples were measured and analyzed on a flow cytometer (3-laser, 10-color; SONY SA3800) using the appropriate mAbs. The antibodies used for cell surface staining included purified rat anti-mouse CD16/CD32 (553142; Mouse BD Fc Block™, BD Pharmingen™), CD4 monoclonal antibody (T helper cell marker, 17-0042-82; RM4-5, APC, eBioscience, San Diego, CA, USA), and rat IgG2a kappa isotype control (17-4321-81; eBR2a, APC, eBioscience), while intracellular staining was performed with PE-Cy™7 rat anti-mouse IL-4 (560699; BD Pharmingen™), PE-Cy™7 rat IgG1κ isotype control (557645; BD Pharmingen™), anti-iNOS-PE cyanine7 (25-5920-82), and anti-arginase 1-PerCP-eFluor 710 (46-3697-82; eBioscience). Additionally, the Intracellular Fixation and Permeabilization Buffer (BD Cytofix/Cytoperm Plus Kit with BD GolgiPlug, 555028; BD Pharmingen™) was used. The experiment was set up according to the recommendations of BD Pharmingen. During sample gating, cells were gated against LLN. The LLN gate determined CD4⁺ cells. IL-4⁺ T cell expression was determined from the gated population.