Mouse NK cells were isolated from fresh BALB/c or C57BL/6 spleens 18 h after poly I:C injection (100 μg intraperitoneally). After red cell lysis, NK cells were purified with MagniSort Mouse NK cell Enrichment Kit (Invitrogen), followed by CD49b MACS-sorting (Miltenyi). This cell population was highly selected for NK cells with a purity of >9%. Human NK cells from PBMCs were purchased from StemCell Technologies containing >99% NK cells.
NK cell killing assays were performed on the XCelligence SP platform (ACEA BioSciences). 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma-Aldrich) and 4 × 105 WT, B2m−/−Ciita−/−, or B2m−/−Ciita−/− Cd47 tg miECs or WT, B2M−/−CIITA−/− or B2M−/−CIITA−/− CD47 tg hiECs were plated in 100 μl cell-specific media containing 1 ng ml−1 mouse or human IL-2 (Peprotech). After the Cell Index value reached 0.7, NK cells were added with an effector cell / target cell (E/T) ratio of 0.5/1, 0.8/1 or 1/1. As a negative control, cell treated with 2% Triton X100 was used. Some wells were pretreated with mouse Cd47 or human CD47-blocking antibody (BioXCell) with 10 μg ml−1 media for 2 h. Data were standardized and analyzed with the RTCA software (ACEA).
NK cell killing assays were performed on the XCelligence SP platform (ACEA BioSciences). 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma-Aldrich) and 4 × 105 WT, B2m−/−Ciita−/−, or B2m−/−Ciita−/− Cd47 tg miECs or WT, B2M−/−CIITA−/− or B2M−/−CIITA−/− CD47 tg hiECs were plated in 100 μl cell-specific media containing 1 ng ml−1 mouse or human IL-2 (Peprotech). After the Cell Index value reached 0.7, NK cells were added with an effector cell / target cell (E/T) ratio of 0.5/1, 0.8/1 or 1/1. As a negative control, cell treated with 2% Triton X100 was used. Some wells were pretreated with mouse Cd47 or human CD47-blocking antibody (BioXCell) with 10 μg ml−1 media for 2 h. Data were standardized and analyzed with the RTCA software (ACEA).