Human foxp3 staining kit

We analyzed the expression of cell surface markers by flow cytometry using FACS Aria II flow cytometer (BD Biosciences) and FlowJo version 7.6.5 software (FlowJo). hiPS-Chons, hPCs, and hPBMCs were stained with anti-human antibodies at 4°C for 30 min. The antibodies used are described in Table 2. HLA expression was also analyzed after treating the cells with 25 ng/mL interferon γ (IFNγ) (Sigma) for 72 h. Isotype control antibodies were used (Table 2). We also, respectively, analyzed T cell proliferation and regulatory T cell population with flow cytometer using the CellTrace^™ Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific) and the Human FOXP3 Staining Kit (BD Pharmingen^™) as described below.

Frozen peripheral blood mononuclear cells were thawed and stained with a master mix of the different antibodies (Table 4) for 15 minute at 4°C in the dark. Afterwards, the wells were washed twice and resuspended in a fluorescence‐activated cell sorting (FACS) buffer. For FoxP3 staining, a human FoxP3 staining kit (BD Pharmingen, Franklin Lakes, NJ) was used according to the manufacturer's instructions. Briefly, cells were first stained with surface antibodies, and then cells were fixed and permeabilized with 2 different buffers. A FoxP3 antibody was then added to the cells, and they were incubated for 30 minutes at room temperature in the dark. When it was necessary, cytotoxic T lymphocyte antigen 4 (CTLA4) and latency‐associated peptide–transforming growth factor β (LAP‐TGFβ) were added to the intracellular antibody master mix to also intracellularly stain those markers. After they were washed twice and transferred to FACS tubes, the cells were placed in a BD LSRII. The acquiring software was FACSDiva (BD Biosciences, Franklin Lakes, NJ). To set the gating for each population, fluorescence minus 1 controls were additionally acquired for each fluorochrome. The analysis was performed with FlowJo (TreeStar, Inc., Ashland, OR). A representative staining and gating strategy is shown in Fig. 1A.

2 × 10⁵ hPBMCs were cocultured with one hiPS-Cart or 1 × 10⁵ mitomycin C-treated hiPS-Chons in the presence of 5 ng/mL IL2 and 1% PHA-M for 96 h. The proliferation of CD4⁺ T cells was detected by flow cytometry analysis using the CFSE Kit. CFSE was added before coculture. hPBMCs cultured in the absence of hiPS-Carts or hiPS-Chons were used as control.
2 × 10⁵ hPBMCs were cocultured with one hiPS-Cart in the absence of 5 ng/mL IL2 and 1% PHA-M for 96 h. The populations of regulatory T cells were measured by flow cytometry analysis using anti-CD4 and anti-CD25 antibodies and the Human FOXP3 Staining Kit (BD Pharmingen).