Detecting NKT-like Cell Functions

The functions of NKT-like cells in PBMCs were detected after coincubation with the K562 cell line or PMA/ionomycin. Because NKT-like cells share some features with NK cells, they can recognize HLA class I-negative cell line K562 and their functions can be detected through coculture with K562 [10 , 11 (link)]. PBMCs were coincubated for six hours with K562 cell line at an E : T ratio of 5 : 1. Meanwhile, PBMCs were stimulated with PMA (Sigma, Cat. No. P-8139, USA) and ionomycin (Sigma, Cat. No. I-0634, USA) in final concentrations of 50 ng/mL and 1 μM, respectively. PE-conjugated anti-CD107a (BD Biosciences, USA) and monensin (Becton-Dickinson, USA) were added to all incubated samples. Then cells were stained with both Percp-conjugated anti-CD3 and PE-cy7-conjugated anti-CD56 (BD Biosciences, USA). The cells were made permeable using Perm/Wash (Becton-Dickinson, USA) for 10 minutes, stained with FITC-conjugated anti-IFN-γ (BD Biosciences, USA) for 30 minutes at 4°C, washed, and then fixed in 1% formaldehyde. NKT-like cell populations were defined by dual-positive expressions of CD3 and CD56 molecules. The frequency of IFN-γ and CD107a expression in NKT-like cells were quantified by multicolor flow cytometry (Figure 1).