Percp cy5.5 conjugated ifnγ

CD14+ monocytes (1 × 10⁵) were cocultured with sorted Treg cells or Teff cells for 3 days at a 1:1 ratio in culture medium containing 100 ng/ml soluble anti‐CD3 monoclonal antibodies (mAb) (OKT‐3; Janssen‐Cilag) in 96‐well U‐bottomed culture plates at 37°C in an atmosphere of 5% CO_2. When indicated, 100 ng/ml lipopolysaccharide (LPS; Sigma) was added on day 0. On day 3, the cells were stimulated with PMA/ionomycin for 6 hours, with GolgiStop present for the last 3 hours, followed by surface staining for Pacific Blue–conjugated CD2 (BioLegend) and APC/Cy7‐conjugated CD14. Cells were fixed with 2% paraformaldehyde and intracellularly stained for PE‐conjugated IL‐17A, PerCP/Cy5.5‐conjugated IFNγ, APC‐conjugated TNF, and Alexa Fluor 488–conjugated IL‐10 (all from BioLegend) using 0.5% saponin.

The frequency of Th17 and Tregs in CD4+ T cells in peripheral blood were determined with the use of flow cytometry as described previously with some modifications (Chi et al., 2007 (link), 2010 (link)). Briefly, 200 μl of fresh peripheral blood obtained from caudal vein of each rat. The red blood cells were lysed and washed with PBS. Cells were initially stained extracellularly with use of phycoerythrin (PE) anti-human CD4 (eBioscience, United States) at 4°C for 20 min. Subsequently, cells were fixed and permeabilized, and stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, United States) for Th17 detection and PerCPCy5.5-conjugated IFN-γ (BioLegend, United States) for Th1 detection. For Tregs, the CD4-PE cells were stained with FITC-conjugated CD25 (BioLegend, United States). Flow cytometric analysis was performed with use of a fluorescence-activated cell sorter Calibur cytometer. Data were analyzed with CellQuest software (Becton Dickinson, United States).