Permeabilization buffer 1x

After the co-culture, we determined CD107a, TNF-α, IFN-γ and granzyme B (BioLegend) by FC. First, NK-92 cells were stain for viability using LIVE/DEAD Fixable Near-IR. After that NK-92 cells were harvested and resuspended in PBS, stained with CD56-PE, and incubated in the dark for 30 min at room temperature. Afterward, the cells were washed and fixed with fixation buffer (BioLegend) for 15 min. Then, the cells were washed and permeabilized with permeabilization buffer 1X (BioLegend), we added TNF-γ-PE-Cy7, granzyme B-AF647, or IFN-α-PE-Cy7, and the cells were incubated in the dark for 30 min. Then, the cells were washed with PBS, fixed with paraformaldehyde 1 %, and analyzed by FC. An appropriate isotype and Fluorescence Minus One (FMO) controls were utilized to adjust for background fluorescence, and results are reported as the % of expression. For each sample, at least 10,000 events were acquired in a FACSAria I cell sorter (BD Biosciences). Data were processed with FlowJo ver. X.0.7 software (Tree Star, Inc.).

Expression of CD163 and TLR was assessed by FC. Briefly, all cells in the different experimental groups were detached, washed twice with PBS, and resuspended in PBS. Then, we blocked human Fc receptors (FcR) using Fc Receptor Blocking Solution (BioLegend, San Diego, CA, USA) for 10 min prior to staining with antibodies. After that, the cells were incubated with antihuman CD163-APC antibody (BioLegend) for 30 min at 4°C. Subsequently, the cells were washed and permeabilized with permeabilization buffer 1X (BioLegend), and we added antihuman TLR-3-Fluorescein isothiocynate (FITC) antibody (Abcam, Cambridge, UK) or antihuman TLR-7-FITC antibody (Abcam) or antihuman TLR-9-FITC antibody (Abcam) for 30 min at 4°C. Then, the cells were washed with PBS, fixed with paraformaldehyde 1%, and analyzed by FC. An appropriate isotype control was utilized to adjust for background fluorescence, and results are reported as the % of expression or as the geometric mean fluorescence intensity (MFI). For each sample, at least 10,000 events were acquired in a FACSAria I cell sorter (BD Biosciences). Data were processed with FACSDiva software (BD Biosciences).