Anti foxp3 af488

Primary unconjugated antibodies used in this study were anti-human CD1a clone OKT6 culture supernatant (from American Type Culture Collection), anti-human HLA-I clone W6/32, anti-human DC-SIGN (27 (link)) (a gift from Dr. Angel Corbí). ICAM-I IgG supernatant (HV5/3) and VCAM-I supernatant, a gift from Dr. Sánchez-Madrid. Fluorescein-coupled polyclonal goat anti-mouse IgG (H + L; Caltag Laboratories) was used as the secondary antibody in FACS analysis. The following conjugated antibodies were used: anti-human CD1d-PE, anti-human HLA-DR-PE, anti-human CD80-PE, anti-human CD86-FITC, anti-human CD25-APC, anti-human CD83-APC (all from BD Biosciences), anti-human iNKT-PE (Miltenyi Biotec), and anti-human CD8-PECy7 (Beckman Coulter), anti IFNγ-AF488 (BD Biosciences), anti FoxP3-AF488 (Biolegend). Flow cytometry was performed following standard protocols on a BD FACSCalibur cytometer. Graph bars summarizing flow cytometry data are expressed as relative mean fluorescence intensity (MFI), which is defined as the MFI of any sample divided by the MFI of the immature dendritic cells (iDCs).
For intracellular staining, cells were fixed with PBS-PFA 4% for 20 min, then, the cells were washed twice in PBS containing 0.1% saponin (Sigma-Aldrich), incubated with the desired antibodies in 100 ml of PBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer.

The following antibodies with their isotype controls were purchased from Biolegend: anti-CD4-BV510, anti-CD3-APC-cy7, anti-IL-17-PE, anti-CD45-PE-cy5.5, anti-IL-21-PE, Anti-IL-21R-PE, anti-CD25-BV421, and anti-Foxp3-AF488. For intracellular staining, the cells were incubated with R10 medium, which is constituted by RPMI1640, 100 ug/mL streptomycin, 100 U/mL penicillin, and 2 mm glutamine, coupled with 50 ng/mL phorbolmyristate acetate (PMA, Sigma, USA), 1 µg/mL ionomycin (Sigma, USA) for 2 h; then 0.7 µL/mL GolgiStop™(BD Biosciences, USA) was added to the cells and incubated for another 3 h. For surface staining, the cells were washed with staining buffer and incubated with fluorescence-conjugated antibodies for 15 min. Then, the cells were washed two times with staining buffer, fixed with fixation buffer for 20 min, permeabilized, and stained with fluorescence-conjugated antibodies for 20 min. The data acquired using a FACS Canto-II flow cytometer (BD Biosciences) were analyzed with Flow Jo software (Tree Star).