C2 confocal microscope

Cells were plated onto coverslips in a 24-well tissue culture plate. They were serum-starved in Opti-MEM Reduced-Serum Medium for 24 hours prior to fixation in 4% paraformaldehyde. To permeabilize cell membranes, coverslips were immersed in 0.1% Triton-X 100 for 5 minutes prior to blocking. Coverslips were blocked in 4% NGS for 30 mins before addition of primary antibodi(es) at 4°C overnight. Secondary antibody was applied for 1 hour, then coverslips were co-stained with DAPI for 15 min. They were sealed to glass slides with ProLong Gold Antifade Mountant. Images were acquired on a Nikon C2 Confocal Microscope. Antibodies and concentrations for immunocytochemistry are listed in Supplementary Table 3.
For analysis of primary ciliation after plasmid transfection, immunocytochemistry slides were blinded to the experimentalist. Z-stack images were then taken on a Nikon C2 Confocal Microscope at 60x magnification. Cilia were quantified by hand-counting these images, with 70-130 cells per replicate per condition depending on the confluency of cells in each image. The transfection experiment and cell counting were repeated (n=2 or n=3) for each condition.

One day before the assay, NK cells were grown in culture in NK cell media supplemented with IL-2 (50 IU/mL) or IL-15 (10 ng/mL) for 12 to 18 hours, and neuroblastoma cells were plated at a density of 50,000 cells per well in 4-well glass bottom chamber slides (Ibidi). On the day of the assay, neuroblastoma cells were labeled with calcein-AM, and NK cells were labeled with CellTracker Orange (Thermo Fisher Scientific), according to manufacturer instructions. Hu14.18K322A (10 μg/mL) was added to the wells for 1 hour to allow binding to tumor cells. Dye-labeled NK cells were then added to the wells containing neuroblastoma cells. A C2 Nikon confocal microscope (Nikon) was used to perform live-cell imaging at a magnitude of 10X for 12 hours. Five different areas were imaged per condition. To quantify the imaging data, we extracted the green (8 bit) and red (16 bit) channels and segmented the green and red signal with FIJI software (12 (link)) and Weka Trainable Segmentation plug-in (13 ). We converted the segmented images into a binary mask and applied the 3D ROI manager plug-in to calculate contact area in μm and signal intensity (14 (link)).

All images were acquired on the same day on a C2 Nikon confocal microscope to limit laser intensity variations with a 60x oil objective, NA = 1.4. Stacks of 200 nm Z-steps were acquired from the surface of the slice to 15–20 um depth on the layer 2/3 of the somatosensory cortex, with a zoom 2, X-Y resolution: 100 nm. Images were deconvoluted using NIS element (Nikon) automatic deconvolution. Post-processed images were 3D-segmented using the toolbox Iterative Thresholding in ImageJ using the following parameters (min threshold: 20, Criteria method: Elongation, Segment Result: best, Threshold method STEP, Value method: 20). 3D colocalizations were analyzed in Fiji (ImageJ) using the Distance Analysis toolbox: DiAna to determine the percentage of JB2-Cy3 puncta or IgG-puncta colocalizing with either presynaptic marker VGLUT1 or postsynaptic marker PSD95 [21 (link)]. For the localization on dendritic compartments on Thy1-YFP slices, stacks were acquired and deconvoluted the same way as the immunostaining images. A 3D mask surrounding the YFP dendritic signal is used to easily see the puncta near the dendrites. Then puncta were assigned to one of the three defined compartments: head spines, spine neck, and dendritic shaft, and normalized in percentage for each neuron.

Confocal images of immunostained neurons were obtained with a Zeiss LSM5 Pascal confocal microscope for two channel images and with a C2+ Nikon confocal microscope for three channel images. Images of neurons were taken using the 63x (Zeiss) or 60x (Nikon) oil-immersion objective (NA = 1.4) as z-series of 5–10 images, averaged 2 times, taken at 0.37 μm intervals, with 1024×1024 pixel resolution at ~140–150 nm/pixel. Detector gain and offset were adjusted in the channel of cell fill (GFP or mCherry) to include all spines and enhance edge detection.
For confocal imaging of N2A cells, images were taken using a 60x (Nikon) oil-immersion objective (NA = 1.4) as z-series of 5–7 images, summed 2 times, taken at 1 μm intervals with 512×512 pixel resolution at ~140nm/pixel.