Pe anti mouse il 4

Both human and mouse CD4⁺ T cells were treated with a leukocyte activation cocktail and incubated at 37 °C with 5% CO2 for 6 h before being harvested for flow cytometry analysis. After the 6-h incubation, the cells were stained with a cell viability marker (Fixable Viability Stain 510 antibody, BD Pharmingen). Subsequently, the cells were treated with surface markers BB515-anti-human-CD4 (BD Pharmingen) and FITC-anti-mouse-CD4 (eBioscience) antibodies for staining. After staining, the cells were fixed and permeabilized with the Cytofix/Cytoperm Soln Kit (BD Pharmingen). Following the cells underwent intracellular staining with the following antibodies: BV421-anti-human-IL-4, PE-Cy [7 (link)]-anti-human-IFN-γ, PE-anti-mouse-IL-4 (all from BD Pharmingen), and PerCP/Cyanine 5.5-anti-mouse-IFN-γ (Biolegend) in permeabilization buffer. Isotype controls were employed in the control group for comparison purposes. Flow cytometry analysis was performed using FACS CantoⅡ (Becton Dickinson), and the data were analyzed using FlowJo version X software.