Fv1200 mpe laser scanning confocal microscope

Immunofluorescence studies were conducted according to standard procedures, as described elsewhere [47 (link)]. Briefly, KNS42 cells were plated on glass coverslips and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Fixed cultures were permeabilized and blocked for 30 min with 5% donkey serum (DS) and 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MI, USA) in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MI, USA). Cells were then incubated overnight with anti-Notch2 antibody (#HPA048743, Sigma-Aldrich, St. Louis, MI, USA). Secondary antibody conjugated with Alexa Fluor 594 (Thermo Fisher Scientific) was diluted 1:200 in PBS with 5% DS and incubated with the specimens for 1 h at RT. Nuclei were counterstained with Hoechst reagent. After washing, slides were mounted using anti-fade reagent (Dako Fluorescence Mounting Medium, Carpinteria, CA, USA). Images were acquired using a FV1200 MPE laser scanning confocal microscope (Olympus, Shinjuku, Tokyo, Japan) with a UPlanSAPO 20×/0.75 NA objective. Imaris 8.1 software (Bitplane, Zürich, Switzerland) was used for image processing.

For chemokine stimulation, we allow primary NK cells to adhere on poly‐l‐lysine‐coated multichamber slides in the presence of CXCL12 for 30 min at 37°C. For analysis of CRBN at NK cell IS, primary NK cells or NK‐92 cells were incubated with K562 target cells in a 1:1 ratio for 7 min a 37°C. Cells were resuspended, allowed to adhere and fixed as previously described [50 (link)], stained with mouse anti‐perforin Ab, Phalloidin‐FITC, and rabbit anti‐CRBN Ab, followed by Alexa Fluor 647–conjugated goat anti‐mouse antibody and Alexa Fluor 594–conjugated goat anti‐rabbit antibody. The coverslips were mounted with SlowFade Gold reagent (Thermo Fisher Scientific), acquired with an FV1200 MPE laser‐scanning confocal microscope (Olympus Life Sciences), and processed with ImageJ software. To determine the percentage of fluorescence of CRBN at IS and the percentage of mature or immature synapse, a polarized region was defined as a triangular area with its tips located at the edge points of the NK‐target cell interface and the center of the NK cell. Granules containing perforin were considered polarized if 70% of granules were localized within this area. The amount of CRBN that localizes to the IS upon conjugation was calculated by analyzing the fluorescence intensity in the polarized region out of total CRBN fluorescence intensity in NK cells.