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Phenotype Analysis and Intracellular Cytokine Staining (ICS) Fresh isolated human PBMCs after stimulating with or without L-Cn (20 mg/mL) for 72 h in the presence of anti-CD3 (5 g/mL, Biolegend) and anti-CD28 (5 g/mL, Biolegend) were stained with fluorescence phenotype antibodies at 4C for 30 minutes and analyzed on a BD FACS-Canto II or Aria III flow cytometer (BD Bioscience)