Anti mouse foxp3 staining kit

PBL and peritoneal macrophages of mice were washed with phosphate-buffered saline (PBS) and stained with PerCP-eFluor-labeled anti-mouse CD40, eFluor 660-labeled anti-mouse CD83, PE-Cyanine7-labeled anti-mouse CD80, and FITC-labeled anti-mouse CD86 (eBioscience, San Diego, CA, USA) at 4°C for 30 min in the dark. For identification of regulatory T cells (Treg cells), isolated PBL was stained with PE-Cyanine7-labeled anti-mouse CD4 and PE-labeled anti-mouse CD25 for 30 min at 4°C in the dark. For intranuclear detection of Foxp3, an anti-mouse Foxp3 staining kit (eBioscience, San Diego, CA, USA) was used, according to the manufacturer's instructions. Briefly, cells were fixed using Fix/Perm buffer and, after washing with 1x permeabilization buffer, were incubated with PE-Cyanine5-labeled anti-mouse Foxp3 Ab for 30 min at 4°C in the dark. Stained cells were analyzed by a FACS Aria III flow cytometer (Becton Dickinson, San Jose, CA, USA) with ≥10,000 gated cells.

Individual cell suspensions were pooled from heart and spleen. Cells were stained with the following mAbs (eBioscience Inc., San Diego, CA, USA) diluted in 1% FBS in PBS: CD4, CD8, CD11b, CD11c, CD19, Gr-1, Ly6G, Ly6C, CD86, F4/80, PDCA-1, Tim-3, CD25, and Foxp3. For intracellular staining, cells were fixed and permeabilized using fixation buffer and permeabilization solution or an anti-mouse Foxp3 staining kit (eBioscience Inc.). Cell fluorescence was measured using FACS and data was analyzed using Cell Quest software (BD Biosciences, San Jose, CA, USA).

To identify regulatory T (Treg) cells, isolated PBL were stained with anti-mouse CD4 and anti-mouse CD25. To detect intranuclear Foxp3, an anti-mouse Foxp3 staining kit (eBioscience, San Diego, CA, USA) was used according to the manufacturer’s instructions. Briefly, the cells were fixed using Fix/Perm buffer, washed with 1x permeation buffer, and incubated with anti-Foxp3 antibody. Then, more than 10,000 cells were analyzed by flow cytometry (FACS Aria III, Becton Dickinson, CA, USA).