Anti mouse cd25 allophycocyanin apc

The surface staining followed routine procedures (8 (link), 30 (link)), and the intracellular staining was performed by use of the Foxp3 staining kit (eBioscience), according to the manufacturer’s instructions. LPL suspensions were stained for surface molecules by incubating with anti-mouse CD4-FITC (eBioscience), anti-mouse CD25-allophycocyanin (APC) (eBioscience), and rat IgG2a isotype control-phycoerythrin (PE) (eBioscience) on ice for 20 min and then washed with PBS buffer. Fixable viability dye (FVD)-eFluor 450 (eBioscience) was used to exclude dead cells. Cells were then fixed in fixation and permeabilization working solution for 30 min, followed by washing and resuspension in permeabilization buffer prior to staining with anti-mouse Foxp3-PE antibody for at least 30 min on ice. Flow cytometric analysis was performed on an LSR Fortessa analyzer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

Cell pellets were prepared from spleen tissues. The regulatory T cell populations were examined using anti-mouse CD4–peridin chlorophyll protein (perCP) and anti-mouse CD25-allophycocyanin (APC) (eBioscience); then, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For Th17 cell analysis, before FACs staining, the cells were stimulated with 25 ng/mL phosphomolybdic acid (Sigma-Aldrich, St. Louis, MO, USA), 250 ng/mL ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences, San Diego, CA, USA) in 5% CO₂ at 37°C for 4 hours. The cells were stained with anti-mouse CD4 PerCP, and then with an anti-mouse IL-17 FITC (eBioscience), followed by fixation and permeabilization using a Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The samples were analyzed using a FACSCalibur instrument (BD Pharmingen; BD Biosciences).