Stellaris dmi8 confocal microscope

Laser micro-irradiation was performed on 1 million U2OS cells cultured on glass-bottomed dishes (MatTek Corp, Ashland, MA, USA) infected with MVMp at an MOI of 25 or transfected with 1 µg of NS1-expression vectors for 16–20 h. Cells were sensitized with 3.5 μL of Hoechst dye (ThermoFisher Scientific, Madison, WI, USA) 5 min prior to micro-irradiation. Samples were irradiated using a Leica Stellaris DMI8 confocal microscope using 63× oil objective and 2× digital zoom with a 405 nm laser using 25 percent power at 10 Hz frequency for 2 frames per field of view. Regions of interest (ROIs) were selected across the nucleus (edges of the nuclei were demarcated and visualized by Hoechst staining). Samples were processed for immunofluorescence imaging and analysis (described below) immediately after micro-irradiation.

APAR body imaging was performed on 500,000 U2OS cells plated on glass cover slips and processed as described below. U2OS cells were pre-extracted with CSK Buffer (10 mM PIPES pH 6.8, 100 mM Sodium Chloride, 300 mM Sucrose, 1 mM EGTA, 1 mM Magnesium Chloride) and CSK with 0.5% Triton X-100 for 3 min each before fixation with 4% paraformaldehyde for 10 min at room temperature. Cells were washed in PBS before being permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Samples were blocked with 3% BSA in PBS for 20 min, incubated with the primary antibody diluted in 3% BSA solution for 20 min, washed in PBS, and incubated with secondary antibody (tagged with the appropriate Alexa-Fluor conjugated fluorophores) for 20 min. Samples were washed in PBS and mounted on glass-bottomed dishes or glass slides with DAPI Fluoromount (Southern Biotech, Birmingham, AL, USA). Images were taken and processed on Leica Stellaris DMI8 Confocal microscope with 63× oil objective lens.

Extent of relocalization of NS1 to micro-irradiation sites was quantified by measuring the intensity of NS1 signal along the γH2AX-labeled stripe. Following microirradiation, images were taken and processed on Leica Stellaris DMI8 Confocal microscope with 63× oil objective lens at 1.5× digital zoom. For cells that were NS1-positive by immunofluorescence, the intensity of NS1 signal over the laser micro-irradiated region was quantified using the plot profile tool on FIJI software. Signal intensities were measured at defined intervals from the left end to the right end of the region of interest (ROI) of irradiated nuclei. Values were averaged for 20–40 nuclei in 2–3 biological replicates at each position along the ROI. Quantification of NS1 relocalized to microirradiated sites relative to total NS1 levels in each individual cell was additionally measured by the ratio (NS1 intensity along the microirradiated stripe)/(total NS1 intensity in the nucleus). Images were processed with FIJI and an ROI outlined the nucleus using DAPI channel to measure total intensity of NS1 while another ROI was drawn outlining the microirradiated stripe using the gH2AX channel.