Cd4 phycoerythrin pe

To analyze the cell surface expression of the typical protein markers, adherent cells were incubated with the following anti-human conjugated primary antibodies (BD Biosciences, Franklin Lakes, NJ, USA): CD4-phycoerythrin (PE) (monoclonal, dilution: 0.2 µl/1×10⁶ cells, catalog no.: 565999); CD8-fluorescein isothiocyanate (monoclonal, dilution: 0.2 µl/1×10⁶ cells, catalog no.: 557696); CD56 (monoclonal, dilution: 0.4 µl/1×10⁶ cells, catalog no.: 556647); and HLA-DR-R-PE (monoclonal, dilution: 0.8 µl/1×10⁶ cells, catalog no.: 560943). Unconjugated markers were added to react with anti-mouse PE-conjugated secondary antibody (eBioscience, San Diego, CA, USA; catalog no.: 11-5921; dilution: 1/200). A total of 10,000 labeled cells were analyzed using a BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA, USA) and the data obtained were analyzed using BD FACSDiva software 6.0 (BD Biosciences).

Foxp3^IRES-GFP mice were sacrificed and their spleen, thymi, and lymph nodes removed, and single-cell suspensions were prepared via disruption of organs through a 40-μM filter. CD4⁺ cells were enriched through negative selection using a CD4⁺ T cell isolation kit following the manufacturer’s protocol (Miltenyi Biotec No. 130–104-454). CD4⁺ cells were stained with CD4-phycoerythrin (PE) (BD Biosciences) and CD25-allophycocyanin (APC) (BD Biosciences) flow cytometric antibodies diluted 1:100 in phosphate-buffered saline (PBS); Tcon cells (CD4⁺ CD25⁻) and Treg cells (CD4⁺CD25⁺ GFP⁺) were sorted on a BD FACSAria (University of Chicago Flow Cytometry Core). Sorted populations were >98% pure. Ex vivo–sorted Tcon cells and Treg cells were maintained and expanded in Advanced Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin/streptomycin, 5 μg/mL gentamicin solution, 2 mM L-glutamine, 1× nonessential a.a.s, 10 mM Hepes, human IL-2 (500 U/mL Tcon cells, 2,000 U/mL Treg cells), and CD3/CD28 Dynabeads at a ratio of 1 bead:1 cell (Tcon cells) or 3 beads:1 cell (Treg cells).