Foxp3 transcriptional factor staining buffer set

To determine the percentage of total T cells (CD3), macrophages (F4/80), and NK cells (NK1.1), splenocytes and mesenteric lymphocytes were blocked with anti-CD16/CD32 mAb (Fc block from eBioscience, San Diego, CA) and then multi-stained with fluorescence-conjugated anti-CD3 (T cells), anti-F4/80 (macrophages), and anti-NK1.1 (NK cells) mAbs (all from eBioscience). To determine the T cell proliferation, anti-CD3/anti-CD28-stimulated cells were blocked and then multi-stained with fluorescence-conjugated anti-CD3 and anti-ki-67 (a cellular proliferation marker) mAbs (eBioscience) using the Foxp3/Transcriptional Factor Staining Buffer Set (eBioscience). All antibodies and reagents were from eBioscience. Data acquired on a Mindray BriCyte® E6 flow cytometer (Shenzhen, China) were analyzed using FlowJo 10.0 software (Tree Star).

Cell suspensions were acquired as previously described18 and were stained in flow cytometry buffer (PBS containing 2% foetal calf serum and 2 mmol/L EDTA). To reduce nonspecific binding, cell suspensions were incubated with antibody cocktails containing anti‐CD16/32 antibody. Cells were extracellularly stained in antibody cocktails for 30 minutes at 4°C, apart from stains containing CXCR5 which were incubated at room temperature in the dark for 1 hour. For detection of intracellular cytokines, cells were incubated with 50 ng/mL phorbol myristate acetate, 500 ng/mL ionomycin and 10 μg/mL brefeldin A for 5 hours at 37°C and 5% CO₂. Cells were fixed with 1% paraformaldehyde. For detection of intranuclear transcription factors, cells were fixed and permeabilized using the Foxp3/Transcriptional factor staining buffer set (eBioscience, CA, USA) according to the manufacturer's instructions. Cells were then washed and intracellularly stained at 4°C in permeabilization wash buffer (Biolegend, CA, USA). Flow cytometry data were acquired using an LSRII Fortessa (Becton Dickson, NJ, USA) and analysed using the FlowJo 10 software (FlowJo, OR, USA). Flow cytometry antibodies are listed in Table S1.