Apc cy7 conjugated cd14

PBMCs (2 × 10⁶ cells/ml) were stimulated in RPMI 1640 culture medium containing 20 mMl‐glutamine (Gibco), 1% penicillin–streptomycin (Gibco), and 10% fetal bovine serum (batch no. F9665, lot no. 030M3399; Sigma) for 3 hours with phorbol myristate acetate (PMA) (50 ng/ml; Sigma) and ionomycin (750 ng/ml; Sigma) in the presence of GolgiStop (BD Biosciences), in accordance with the manufacturer's instructions. Surface staining for PE/Cy7‐conjugated CD3, APC/Cy7‐conjugated CD14 (BioLegend), and Pacific Blue–conjugated CD45RO was followed by intracellular staining for PerCP/Cy5.5‐conjugated CD4, PE‐conjugated IL‐17A (BioLegend), FITC‐conjugated TNF (BioLegend), or Alexa Fluor 647–conjugated FoxP3.

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.