The normal healthy controls were age-matched or relatives without detectable mutations in each allele. Briefly, 1 × 106 PBMCs were surface labeled with peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 (SK3, BD Biosciences, San Jose, CA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD25 (2A3, BD Biosciences). The Treg cells were gated from CD4+CD25 high cells and quantified using intra-cellularly labeled phycoerythrin (PE)-conjugated anti-FOXP3 (PCH101; biosciences, San Diego, CA) (37 (link)).
Th17 cells were counted by intracellular staining of CD4+ T cells for IL-17 production after 4 h of stimulation with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO) in the presence of 1 μl/ml GolgiStop (Becton Dickinson, Franklin Lakes, NJ) (38 (link)). After surface staining with PerCP -conjugated anti-CD4, PBMCs were fixed, permeabilized (Cytofix/Cytoperm; Pharmingen), and stained with PE-conjugated anti-IL17A (eBioscience, San Diego, LA) as previously reported (30 (link)).
Data were analyzed using CellQuest™ analysis software (BD Biosciences). The proportion of Treg cells was determined based on CD4+CD25+FOXP3+ and Th17 cells on CD4+IL17+ after non- and stimulation, respectively.
Th17 cells were counted by intracellular staining of CD4+ T cells for IL-17 production after 4 h of stimulation with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO) in the presence of 1 μl/ml GolgiStop (Becton Dickinson, Franklin Lakes, NJ) (38 (link)). After surface staining with PerCP -conjugated anti-CD4, PBMCs were fixed, permeabilized (Cytofix/Cytoperm; Pharmingen), and stained with PE-conjugated anti-IL17A (eBioscience, San Diego, LA) as previously reported (30 (link)).
Data were analyzed using CellQuest™ analysis software (BD Biosciences). The proportion of Treg cells was determined based on CD4+CD25+FOXP3+ and Th17 cells on CD4+IL17+ after non- and stimulation, respectively.
Free full text: Click here
