Allophycocyanin apc conjugated anti human cd25

The following antibodies (to human) were from BD Biosciences (Fremont, CA, USA): fluorescein isothiocyanate conjugated anti-human CD4, phycoerythrin (PE)-conjugated anti-human CD4 and IL-17A, allophycocyanin (APC)-conjugated anti-human CD25, phycoerythrin-Cy7-conjugated anti-human IFN-γ, IL-4, and their isotype-matched control antibodies (mouse IgG1, mouse IgG2a). The following antibodies were from eBioscience (San Diego, CA, USA): PE-Cy7-conjugated Foxp3. Purified anti-human CD3, anti-humanCD28, anti-human IL-6 antibodies and recombinant IL-6 antibodies were all from R&D Systems (Minneapolis, MN, USA). AKT inhibitor (GSK690693), p38/MAPK inhibitor (SB203580) and STAT3 inhibitor (NSC74859) were from Sigma-Aldrich (St. Louis, MO, USA). Human IL-6, TGF-β ELISA kits were from BioLegend (San Diego, CA, USA). The following antibodies (to human) were from Cell Signaling technology (Inc., USA): STAT3, AKT, MAPK, p38, p-ERK1/2, p-MAPK, p-AKT, p-p38. The following antibodies (to human) were from Proteintech (Inc., China): ERK1/2, ERK1/2, p-STAT3, and GAPDH.

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.