Pe cy7 anti mouse cd25

Single-cell preparations with one million cells per 100 ul PBS were stained with antibodies to the following markers: APC/Cy7-anti-mouse CD4 (100413; Biolegend), PE/Cy7-anti-mouse CD25 (102015; Biolegend), APC-anti-mouse B220 (103221; Biolegend), PE-anti-mouse CD45 (103105-50; Biolegend). For intracellular staining, all cells were fixed for 40 min in BD Fix/Perm buffer and then washed in BD Perm/Wash buffer. The cells were then stained with PE-anti-mouse FOXP3 (126403; Biolegend) and FITC-anti-mouse IgA (11-4204-81; Biolegend) for 30 min at 4°C. Stained cells were tested on a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, USA).

Single-cell preparations were stained for Treg cells with surface antibodies to the following markers: APC/Cy7-anti-mouse CD4 (100413; Biolegend), PE/Cy7-anti-mouse CD25 (102015; Biolegend). While the single-cell suspensions used to stain Th cells needed to be incubated with Cell Activation Cocktail (Biolegend, San Diego, USA) in 5% CO2 at 37 °C for 6 h. After stimulation, the cells were stained with anti-CD4. For intracellular staining, all cells were fixed for 40 min in BD Fix/Perm buffer and washed in BD Perm/Wash buffer twice, and then incubated with FITC-anti-mouse IFN-γ (AM0IF01-20; MULTI SCIENCES), PE-anti-mouse IL-17A (AMO1704-20; MULTI SCIENCES), APC-anti-mouse IL-4 (AMOI405-20; MULTI SCIENCES), or PE-anti-mouse FOXP3 (126403; Biolegend) for 30 min at 4 °C. Stained cells were tested on a FACSVerse flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (TreeStar, USA).