Hcx pl apo cs

Cells were fixed in 4% formaldehyde, permeabilized, and blocked in BSA. DNA was stained with DAPI. For γ-H2AX staining, cells were processed as described [7 (link)]. Images were taken with an SP8 Leica laser scanning confocal microscope and software (Leica Microsystems GmbH, Wetzlar, Germany) with HCXPL APO CS 40× or 63× objective lenses. Nuclear γ-H2AX fluorescence signal was quantified by calculating the pixel intensity in single cell nuclei relative to the nucleus area. At least 200 randomly selected cells per condition were quantified using ImageJ.

Samples were analysed with Leica TCS-SP5 Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany) using HCX PL APO CS 63.0 × 1.20 WATER UV lens, 1.33 refraction index. Scanner settings were calibrated with PinHole (m): 133.6 μm; PinHole (airy): 1.2; Zoom: 1.7. Images were taken step sizing the size-depth, optimizing the number of section by halving the numbers provided by the system. Hardware was set to have all lasers active (405 Diode, UV; Argon, Visible; DPSS 561, Visible; and HeNe 633, Visible) with Argon, Visible at 29%. In order to distinguish nuclei fluorescence from antibody-labelled plasma membrane extrusions, DAPI was exited using pre-set DAPI parameters, calibrating Laser Line UV (405) at 27% and all other Laser Line at 0%. Emission PMT was calibrated between 417 and 496 nm, Gain: 693 nm, Offset: 0, Transmission: 504, Offset: 0. To detect DyLight Antibody with excitation/emission rate 493/518 nm, pre-set FITC parameters were adopted, calibrating Laser Line visible (488) at 76% and all other Laser Line at 0%. Emission PMT was calibrated between 500 and 560 nm, Gain: 808 nm, Offset: 0, Transmission: inactive. All parameters were adjusted by the company-provided software (Leica Microsystems LAS AF TCS MP5). Images were analysed using the same software and elaborated using ImageJ 1.42 (available from public domain at http://rsbweb.nih.gov/ij/index.html).

The 3D microstructures and 3D fluidics in the channels were observed with a scanning electron microscope (SEM, TM-1000, Hitachi, Tokyo, Japan) and a laser confocal microscope (TCS-STED-CW, Leica Microsystems, Wetzlar, Germany). Aqueous solutions of 0.5 mM fluorescein sodium salt (Sigma-Aldrich, Tokyo, Japan) and 0.1 mM rhodamine B (Sigma-Aldrich, Tokyo, Japan) were used to visualize the 3D flow and observed with a 442-nm excitation laser and a 10×/0.40 lens (HCX PL APO CS, Leica Microsystems GmbH, Wetslar, Germany). Stacks of each confocal X-Y scan of 1024 × 1024 pixels were collected with a step of 0.49 µm in the Z direction. Z-series images were loaded in to the imaging software (LAS AF, Leica Microsystems GmbH, Wetslar, Germany) and made into vertical cross-sectional images.

Cells were fixed in 4% formaldehyde, permeabilized and blocked in BSA. F-actin was stained using Phalloidin eFluor-570 (eBioscience, Paisley, UK) and DNA was stained with DAPI. Images were taken with a SP8 Leica laser scanning confocal microscope and software (Leica Microsystems GmbH, Wetzlar, Germany). Backscattered light (reflectance) was collected to image the matrix surrounding cells. For cMyc and γ-H2AX staining, cells were processed as described in [45 (link)]. Images were taken with a SP8 Leica laser scanning confocal microscope and software (Leica Microsystems GmbH) with HCXPL APO CS 40x or 63x objective lenses. Nuclear cMyc and γ-H2AX fluorescence signal was quantified by calculating the pixel intensity in single cell nuclei relative to the nucleus area. At least 200 randomly selected cells per condition were quantified using ImageJ.

For iFRAP, cells were seeded on coverslips and imaged on a Leica SP8 microscope using a 40/1.25 NA HCX PL APO CS oil immersion lens (Leica Microsystems) at 37 °C and 5% CO_2. The entire nucleus was bleached with 20% 488 nm laser power except for two non-bleached areas where fluorescence signal was monitored over time: LUD and a non-damaged nuclear area. Background signal was measured in the cytoplasm. iFRAP curves were obtained after background correction and normalization of the damaged area to the fluorescence levels of the pre-damage conditions. The non-damaged area served as internal control and was not plotted. To obtain the half-time of protein residence in the LUD the non-linear regression fitted to one-phase exponential decay analysis was applied to the iFRAP curves, using Graph Pad Prism version 9 for Windows (GraphPad Software, La Jolla California USA).

Cells were grown on coverslips before transfection with the indicated plasmid DNA. 24 hours after transfection, cells were washed with phosphate buffered saline (PBS) (pH7.2), fixed with 4% paraformaldehyde for 10 min at room temperature, followed by a 10 min permeabilization with 0.3% TX-100 at room temperature. Cells were then blocked with 5% BSA (bovine serum albumin) in PBS, and further incubated for 2 hours with primary antibodies (anti-ORF1p (1:1000 dilution), anti-γH2AX (1:1000 dilution) or anti-G3BP1 (1:1000)) at room temperature. Alexa fluor 555-conjugated donkey anti-rabbit antibody (A-21428) and Alexa fluor 647-conjugated donkey anti-mouse antibody (A-21236) were used as secondary antibodies. Confocal images were recorded with a Leica TCS SP5 (Leica Microsystems) mounted on an inverted microscope (DMI6000; Leica Microsystems), with an oil immersion 63x/NA1.4 objective len (HCX PL APO CS; Leica Microsystems).