Pe conjugated anti mouse foxp3

Spleen were obtained from the mice and processed to achieve single cell suspensions by pressing the spleen through a 40-μm cell strainer in PBS. The red blood cells in splenic suspensions were lysed with 0.75% NH₄Cl and Tris buffer (0.02%) (pH = 7.4) for 5 min. For surface staining, splenocytes were first incubated with PE-Cy5-conjugated anti–mouse CD4 and APC-conjugated anti–mouse CD25 (ebioscience) for 30 min at 4 °C. Cells were further fixed and permeabilized with Cytofix/Cytoperm solution (Catalog, 85-00-5523-00, ebioscience). Then, cells were stained with PE-conjugated anti-mouse Foxp3 (ebioscience), Brilliant Violet 421™-conjugated anti-mouse IL-10 (Biolegend), and Brilliant Violet 421™-conjugated anti-mouse TGF-β (Biolegend) for 30 min at 4 °C. Cells were analyzed using a BD FACSort flow cytometer (BD Biosciences) by gating CD4+ cells and examining the percentage of CD25+Foxp3+ Treg as well as IL10+/TGF-β + Treg. A minimum of 30,000 events was acquired for each sample. Isotype-matched control antibodies were used to determine the cut-off between negative and positive populations. Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

For Treg detection, cells were first stained with FITC-conjugated anti-mouse CD45 (BD, United States), PE-CY-7-conjugated anti-mouse CD4 (BD, United States), and APC-conjugated anti-mouse CD25 (BD, United States). For Th17 cell detection, cells were stimulated with Cell Stimulation Cocktail in culture medium for 4 h before surface staining using FITC-conjugated anti-mouse CD45 and PE-CY-7-conjugated anti-mouse CD4. After surface staining, cells were fixed and permeabilized with fixation/permeabilization buffer, followed by staining with PE-conjugated anti-mouse Foxp3 (eBIOSCIENCE, United States) for Tregs and PE-conjugated anti-mouse IL-17A (BD, United States) for Th17 cells. For dendritic cells detection, cells were stained with FITC-conjugated anti-mouse CD11c (BD, United States). Cells were analyzed on a FACS flow cytometer (CytoFLEX, Beckman, United States and BD Biosciences, United States).

To evaluate regulatory the Treg percentage, single-cell suspensions of splenocytes were prepared according to the method described by Mo et al. (19 (link)). Briefly, resected spleens were ground and filtered through a 200-mesh filter cloth. Red blood cells in the generated cell pellet were cleaved using an erythrocyte lysate (Beyotime, China), and 5 × 10⁶ cells were suspended in Roswell Park Memorial Institute (RPMI)-1640 medium. The cells were then stained with conjugated antibodies including FITC-conjugated anti-mouse CD4, APC-conjugated anti-mouse CD25, and PE-conjugated anti-mouse Foxp3, using a mouse Treg-staining Kit (eBioscience). Finally, they were analyzed using a BD Biosciences FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).