Biorevo bz 9000 fluorescence microscope

Cells were fixed in 4% paraformaldehyde at 4°C for 15 min. in the presence of a protein-blocking solution consisting of PBS supplemented with 5% normal goat serum (X090710-8, Agilent Technologies Inc., SantaClara, CA, USA). The cells were incubated overnight with anti-TFEB antibody (ab267351, Abcam plc.) in PBS at 4°C. The cells were washed extensively in PBS and incubated at room temperature for 30 min with a anti-rabbit IgG (H + L) antibody tagged with Alexa FluorTM 488 (Thermo Fisher Scientific, Inc.). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; diluted 1:500, #5748, FUJIFILM Wako Pure Chemical) in PBS at room temperature for 30min. We obtained the fluorescence images using a Biorevo BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). The identification of TFEB migrated to nuclear was performed using ImageJ. First, the multicolor image was separated into TFEB- and DAPI-stained images. These images were converted to binarized images by thresholding, where a foreground pixel was assigned the maximum value of 255 and background pixels were assigned the minimum possible value of 80. The area where the TFEB and DAPI areas overlap is defined as the nuclear TFEB. The percentage of cells in the image with nuclear TFEB was calculated.

Cells were grown on micro cover glass (Matsunami), fixed in 4% paraformaldehyde, permeabilized by 0.1% Triton X-100, and blocked with 3% bovine serum albumin in PBS. Incubation with a series of primary antibody was conducted at room temperature for 1 h and was followed by 1 h incubation at room temperature with secondary antibodies. For secondary antibodies, Alexa^TM 594-conjugated and Alexa^TM 488-conjugated anti-rabbit or -mouse IgG antibodies (Molecular Probes) were used at 1 : 800 dilutions. To observe the nucleus, cells were stained with 2.5 µg/ml diamidino-2-phenylindole (DAPI) or Hoechst 33342 in PBS at the time of antibody staining. Immunofluorescent images were obtained with a BIOREVO BZ9000 fluorescence microscope (Keyence) and an LSM510 inverted confocal microscopy system (Carl Zeiss).

3-dimensional PAH media tissues were generated using a 3D cell culture technique reported previously (Tanaka et al., 2019 (link)). Briefly, trypsinized PASMCs were first incubated in Tris–buffered saline (pH 7.4) containing 0.04 mg/mL Fibronectin (Sigma-Aldrich, St. Louis, MO, United States) and 0.04 mg/mL Gelatin (Wako Pure Chemicals, Osaka, Japan) upon gentle rocking [30 min, room temperature (RT)]. 5.0 × 10⁵ PASMCs were then seeded on cell culture inserts for 24 well plates (0.4 μm, transparent; BD Falcon/Corning, Corning, NY, United States) coated with 0.12 mg/mL Fibronectin. After 3 days of incubation, 3D-PAH media tissues were fixed with 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 20 min, RT) for hematoxylin-eosin staining (Applied Medical Research, Osaka, Japan) and observed under a BioRevo BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

Cells were imaged with a BIOREVO BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan). Images were acquired every 60 or 120 min. During imaging, cells were held in an incubation chamber at 37°C in a humidified atmosphere containing 95% air / 5% CO₂ (Tokai Hit, Fujinomiya, Japan). After irradiation, each cell was monitored until the next mitosis, and the changes in fluorescent colors and their durations were recorded. Green fluorescence intensity within each nucleus was quantitated using the Dynamic Cell Count software (KEYENCE). To adjust the fluorescence intensities among different fields, the background fluorescence intensity of each image was normalized to 1. A common database obtained from 627 cells flash-labeled with EdU immediately after 5 Gy irradiation was obtained for the analysis.