Apc conjugated anti mouse cd25

Splenic lymphocytes from mice were isolated according to a previously reported method [14 (link)], and these cells were stained with APC-conjugated anti-mouse CD25 and FITC-conjugated anti-mouse CD4 or isotypes (eBioscience, USA) for 20 min at 4 °C. These cells were washed twice, fixed, permeabilized and then stained with PE-conjugated anti-mouse Foxp3 for analysis of Treg subpopulations. To detect splenic Th17 cells, splenocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (BioVision, Mountain View, CA, USA), 1 μg/ml ionomycin (Enzo Life Sciences, Farmingdale, NY, USA) and 500 ng/ml monensin (eBscience, San Diego, CA, USA) for 4 h and were stained with FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse IL-17 antibodies. Acquisitions were performed using a FACS Canto II flow cytometer (BD, USA). Data were analyzed based on the percentages of Th17 cells and Treg cells.

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).

Spleen cells were collected; the red blood cells were lysed with buffer containing NH₄Cl, and adjusted to 10⁶ cells/tube. CNS-extracted cells were plated at 5 × 10⁵ cells/well and stimulated with MOG (125 μg/mL) and with C. albicans (5 yeasts/1 cell). After incubation at 37°C for 48 h, cells were collected and stained. Spleen and CNS-extracted cells were blocked with rat serum 1% for 20 min to prevent nonspecific binding via Fc receptor. After Fc blocking, cells were stained with 0.2 μg of PerCP-conjugated anti-mouse CD3 and 0.25 μg of FITC-conjugated anti-mouse CD4 for 20 min at 4°C. Intracellular FoxP3 transcription factor analysis was performed only in spleen samples by using CD3-PercP, CD4-FITC plus 0.13 μg of APC-conjugated anti-mouse CD25 and 0.2 μg of PE-conjugated anti-mouse FoxP3 and staining set (eBiosciences, San Diego, CA, USA) according to manufacturer's instructions. After staining, the cells were washed, resuspended in FACS buffer, and fixed in paraformaldehyde 1%. Analysis was performed using a FACSCanto II (BD) from Bioscience Institute (Botucatu, SP, Brazil) and the data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).