Figure 3 Mechanism of Kupffer cell-mediated regulation of NK cells via a receptor-ligand pathway in PBC mice. (A) NK cells were sorted with immunomagnetic beads. After 1 hour, the morphology of the cells was observed under a microscope. a) 100×, cells were round, b) cells at 200×. (B) Immunofluorescence. F4/80 (green)-labeled Kupffer cells and PI (red)-labeled nuclei were observed with laser confocal microscopy. (C) Flow cytometry detection of NKG2D and RAE-1 expression in NK cells cultured for 36 hours and Kupffer cells cultured for 72 hours. Data are expressed as the mean±SEM, n=3 for each group, *** P<0.001. (D) The upper chamber was withdrawn, and NK-Kupffer cell contact was eliminated. Flow cytometry was applied to measure the expression of NKG2D, RAE-1, and F4/80 after the coculture of NK cells from mice in the PBC and CON groups in Transwell chambers with unstimulated Kupffer cells. Data are expressed as the mean±SEM, n=3 for each group, ** P<0.01. (E). NK cells from mice in the PBC and CON groups were cocultured in Transwell chambers with Kupffer cells stimulated with 10 μg/mL LPS, and the expression levels of NKG2D, RAE-1 and F4/80 were detected by flow cytometry. The data are expressed as the mean±SEM, n=3 for each group, *** P<0.001, * P<0.05, ** P<0.01. (F) The upper chamber was withdrawn, eliminating NK cell-Kupffer cell contact, following which the NK cells in the PBC and CON groups were cocultured with Kupffer cells stimulated with 10 μg/mL LPS in Transwell chambers, and the expression levels of NKG2D, RAE-1 and F4/80 were detected by flow cytometry. The data are expressed as the mean±SEM, n=3 for each group, *** P<0.001. NK – natural killer; PBC – primary biliary cholangitis; NKG2D – natural killer group 2, member D; PI – propidium iodide; RAE-1 – retinoic acid early inducible-1; SEM – standard error of the mean; CON – control; LPS – lipopolysaccharide.