Foxp3 staining kit

Naïve CD4⁺ T cells were isolated from the spleens according to the manufacturer’s instruction (Stem Cell) and stimulated with addition of IL-2 (30U/ml) and TGFβ (15ng/ml) for Tregs or TGFβ (15ng/ml), IL-6 (10ng/ml), anti-mouse IFNγ Ab (5µg/ml) and anti-mouse IL-4 Ab (5µg/ml) for Th17 for 4-5 days. Foxp3 was stained using FoxP3 Staining Kit (BD560131) according to the instruction.

In blood samples from different mice groups, circulating levels of T lymphocytes were evaluated. Cell surface staining was performed using FITC-labeled anti-CD3 or anti-CD8 and PE-labeled anti-CD4 (BD Pharmingen). After cell surface staining, Foxp3 was stained using Foxp3 staining kit (BD Pharmingen) according to manufacture instruction. Flow cytometry analysis was conducted on a FACSCalibur (BD Biosciences) with Cell Quest Pro software.

1×10⁶ PBMCs from pre stimulation, CMVpp65 stimulated and CD25 positive fractions were stained with CD3-APC, CD25-PE, CD4-APC Cy7 and CD8-PerCP then fixed and permeabilized using the FoxP3 staining kit (BD Bioscience) before staining with either FoxP3-Alexa Fluor 488 monoclonal antibody or matched IgG isotype control. Samples were acquired on the FACScan flow cytometer with a minimum of 50,000 CD4+ events recorded.

Th1, Th2 and Th17 frequencies were determined using intracellular staining followed by flow cytometry [24 (link)]. Whole blood (500 μl) was diluted 1:1 with RPMI medium supplemented with 10% fetal bovine serum and 1% antibiotic, and cultured with 50 ng PMA (Sigma, St Louis, MO, USA), 1 μg/ml ionomycin (Sigma, USA) and 10 μg/ml brefeldin-A (Sigma, USA) at 37°C in 5% CO₂ for 6 h. Then, the cells were first surface-stained with anti-CD4-FITC and anti-CD3-APC antibodies, followed by fixation and permeabilization using cytofix/cytoperm kit (BD Biosciences, Franklin Lakes, NJ, USA). The permeabilized cells were then treated with anti-IFNγ-PE, anti-IL-17A-PerCP-Cy5.5 or anti-IL-4-PE intracellular antibodies. The Th1, Th2 and Th17 cells were then counted as CD4⁺/IFNγ⁺ cells CD4⁺/IL-4⁺ cells and CD4⁺/IL-17A⁺ cells, respectively, in the CD3 gate.
For Treg enumeration, Foxp3 staining kit (BD Pharmingen, USA) was used. In brief, dual surface staining for anti-CD4-FITC and anti-CD25-APC, and intracellular staining for anti-Foxp3-PE was done as per the manufacturer’s instruction. The data were acquired using a BD Canto II (BD Biosciences) flow cytometer and FACS Diva software. CD25⁺/Foxp3⁺ cells in CD4⁺ gate were considered as Tregs [24 (link)]. An unstained tube was used as negative control for each specimen.

5 × 10⁶ cells were resuspended in 100 μl staining buffer (1% BSA, 0.1% sodium azide in PBS). Cells were blocked with Fcγ receptor for 15 min and stained for surface antigens with flow cytometry Abs for 30 min at 4 °C. A fixable LIVE/DEAD stain (Invitrogen) was used to measure viability, and FSC/SSC parameters were used to exclude doublets from analysis. For intracellular staining, cells were treated using the Foxp3 Staining Kit (BD) according to the manufacture’s instructions. Cellular fluorescence was determined using a LSRII, or a FACSCalibur flow cytometer (BD Biosciences), and data analyses were performed with FlowJo software (Tree Star). The antibodies used in this study are shown in Supplementary Table 1.