Pe conjugated anti cd107a

For flow cytometry analysis, cells were cultured mono/co-culture spheroids, and then collected and trypsinized to obtain a single-cell suspension. Briefly, cells were washed in FACS buffer (PBS supplemented with 2% FBS) and, stained according to the manufacturer’s instructions in 100 µL FACS buffer supplemented with APC-conjugated anti-EPCAM (1:50, BD Biosciences, San Jose, CA, USA), PerCP-Cy 5.5-conjugated anti-CD56 (1:100, BDBiosciences, USA), PE-conjugated anti-CD107a (1:100, BD, Biosciences, USA) and APC conjugated anti α-SMA (1:2000, R&D Biotechne, Minneapolis, MN, USA). Cells were kept in the dark at RT for 20 min. Live/Dead cell analysis, Annexin-V-FITC (1:100, Cell Signaling Technology, Danvers, MA, USA) and Propidium Iodide-PE (1:10, Cell Signaling Technology, USA) were used. Stained cells were washed twice in DPBS to remove unbound antibodies, resuspended in 200 µL FACS buffer and analyzed in BD FACSCalibur Flow Cytometer (BD Biosciences). An unstained negative control was run to establish the fluorescence gates. First, the cells were gated in an FSC-A (forward scatter) and SSC-A (side scatter) dot plot to eliminate doublets. EPCAM (+) cells were chosen to analyze dead/live ratio in cancer cells. In order to identify CD107a (+) NK-92 cells, the CD56 (+) cell population was gated. All data were then analyzed using FlowJo X software Version 9.

For immunofluorescent analysis of spheroid cultures, cells were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) at RT for 15 min, washed three times with PBS, and blocked with 3% BSA in PBS. Following the blocking step, PE-conjugated anti-CD107a (1:50, BD Pharmingen, San Diego, CA, USA) primary antibody were applied to stain NK cells. Then the cells were washed three times and incubated with DAPI (250 nM, Biolegend, San Diego, CA, USA) at RT for 15 min to observe nuclei morphology. Cells were coverslipped with mounting medium (Dako) then observed under a confocal microscope (Leica). Immunofluorescence of PSC cells was analyzed as described above. Depending on the targeted proteins such as intracellular antigens, cells were permeabilized before blocking with 0.1% Triton X-100 (Sigma) to detect anti-Vimentin (1:1000, Cell Signaling Technology, USA) and anti-α-SMA (1:1000, R&D Biotechne, USA).

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).