Human regulatory t cell staining kit

The cells were routinely fixed with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and stained with antibodies at 18°C. To test for intracellular FoxP3, lymphocytes were fixed and permeabilized using the Human Regulatory T Cell Staining Kit (eBioscience) following the manufacturer's instructions. The cells (at least 10⁴ cells per test) were analyzed with an Epics Elite flow cytometer (Coulter, Marseille, France) in the logarithmic channel of fluorescence. The data were processed with EXPO32 software (Applied Cytometry Systems, Sheffield, UK). The gating strategy and controls for Figures 1–3 are described in Supplemental Figures 2–4.

Human monoclonal Abs against CD4, CD8, Foxp3, CD25, TGF-β, and IL-10 conjugated with different fluorescent dyes were purchased from BD Bioscience (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). In the present study, Tregs from peripheral blood mononuclear cells (PBMCs) or tumor tissues were classified as CD4⁺Foxp3⁺ cells via fluorescence-activated cell sorting (FACS) using a multiplex gating strategy. Transcription factor staining was performed using a human regulatory T cell staining kit (eBioscience) and Foxp3 APC Abs (eBioscience). In brief, the harvested cells were washed and stained with surface phenotypic markers for 20 min on ice. After permeabilization and fixation, the cells were intracellularly stained with Fxop3 ABC Abs, and the positively stained cells were detected using a Beckman Coulter Gallios Flow Cytometer and analyzed using FlowJo V10 software.

Whole blood for analyzing CD4+ T cell cytokines was obtained twice from enrolled patients, at the time of diagnosis and 1‒2 months after first TACE, and was stored at −20 °C. Serum concentrations of CD4+ T cell cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-22, IFN-γ, and TNF-α) were measured by enzyme linked immunosorbent assay (Flowcytomix™ Multiplex) using Multiplex cytometric bead immunoassay (eBioscience, San Diego, CA, USA). Whole blood obtained at baseline was also used to measure Tregs using a Human Regulatory T-cell Staining Kit (eBioscience, San Diego). Peripheral blood mononuclear cells were isolated by centrifugation with Ficoll-Hypaque gradients and incubated with anti-CD4-FITC and anti-CD25-APC monoclonal antibodies for 30 minutes at 4 °C.

For cell surface staining and analysis, 1 × 10⁶ PBMCs were incubated with the specific antibodies (eBiosciences, San Diego, USA) for 30 min at room temperature. The antibodies used included anti-CD3-APC for T cells, anti-CD3-APC and anti-CD4-PE or anti-CD8-FITC for CD4⁺ or CD8⁺ T cells. Cells were sorted by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, USA) and the data was analyzed using CellQuest software (Version 1.22, Becton Dickinson).
For detecting the proportion of CD4⁺CD25⁺ Treg cells, intracellular FoxP3 staining was performed using Human Regulatory T Cell Staining Kit (eBioscience, San Diego, USA) according to the manufacturer’s protocol. Briefly, 1 × 10⁶ PBMCs were labeled with anti-CD4-FITC and anti-CD25-APC, followed by fixation and permeabilization with Cytofix/Cytoperm medium (BD Pharmingen, San Diego, USA). Then PBMC were intracellularly stained with Phycoerythrin (PE)-conjugated anti-Foxp3 or IgG2a isotype control antibodies. The CD25⁺Foxp3⁺ Treg cells were analyzed after gating on the CD4⁺ T cells.

The basic immune-profiling was performed in 5/6 CML patients using flow-cytometry that detected populations of CD4+, CD8+, regulatory T-cells (Tregs) and NK cells before IFN-α initiation and during IFN-α treatment. Peripheral blood samples were stained with anti-CD3 FITC clone MEM-57, anti-CD4 FITC clone MEM-241, anti-CD8 PE clone MEM-31, anti-CD16+CD56+ PE clones LNK-16 and MEM-188, anti-CD4 APC clone MEM-24 and anti-CD8 PerCP clone MEM-31 (Exbio Prague, Czech Republic) using a lyse/no-wash protocol. Minimal acquisition was designated as 10,000 CD3+ events. Intracellular Foxp3 analyses were performed using a Human Regulatory T Cell Staining Kit (eBioscience, San Diego, CA, USA) which contains the Foxp3 PE clone PCH101, CD4 FITC and CD25 APC antibodies plus buffers and controls. The staining protocol was optimized with PBMCs. Fresh Ficoll prepped PBMCs were surface stained with anti- CD25 APC and anti-CD4FITC antibodies and then subsequently with anti Foxp3 PE antibody (intracellular staining). CD25+CD4+Foxp3+ cells were considered as T regulatory cells. Minimal acquisition was designated as 10,000 CD25+ CD4+ events. Analysis of immune competent cell subpopulations was performed using the FACS Diva (BD Biosciences, San Jose, CA, USA) software. Absolute numbers were expressed as the number of cells ×10⁹/L.

The monocyte isolation kit used was Ficoll-Paque Plus (GE Healthcare, USA). CD45 cells were isolated with RosetteSep Human CD45 Depletion Cocktail (Stemcell Technologies, Canada). Other equipment used included the RNA extraction kit, RNeasy Mini Kit (Qiagen, Germany), the QuantiTect Reverse Transcription kit, the Human Regulatory T cell Staining Kit (eBioscience, USA), the LightCycler 480 Real-time PCR system (Roche, Switzerland) and the FACS Calibur flow cytometry system (BD Biosciences, USA).

The expression of CD39 and CD73 in circulating CD4⁺CD25⁺Foxp3⁺ T cells, as Tregs, was evaluated using the Human Regulatory T cell Staining Kit (eBioscience, San Diego, CA, USA), according to the manufacturer’s protocol. Single-cell suspensions were incubated with PerCP-conjugated anti-human CD4 (BD Biosciences), allophycocyanin (APC)-conjugated anti-human CD25 (BD Biosciences), FITC-conjugated anti-human CD39 (BD Biosciences), and PE-cy7-conjugated anti-human CD73 (BD Biosciences) antibodies for 30 min in the dark at 4 °C to stain surface-expressed factors.
After washing with flow cytometry staining buffer, the PBMCs were incubated with 1 ml freshly prepared Foxp3 fixation/permeabilization buffer for 20 min at 4 °C in the dark. Then, the cells were washed twice with 2 ml freshly prepared 1 × permeabilization buffer. Next, the cells were stained using a PE-conjugated anti-human Foxp3 (eBioscience) antibody for 30 min in the dark at 4 °C. After washing twice, the number of Foxp3-positive cells in the CD4⁺CD25⁺ cell gating was evaluated by flow cytometry, and the frequency of Foxp3⁺ Treg cells was expressed as a percentage of the total CD4⁺ CD25⁺ cells.