3d cell explorer

HEI-OC1 cells (2 × 10⁵ cells in 6-well cell culture plate) were incubated with ethanol (0.05% (v/v), 4 h). Further, 3D images were analyzed by 3D Cell Explorer (NANOLIVE, Ecublens, Switzerland). The images were representative images from total 100 cells in ten individual images per group.

HT-1080 cells (80,000 cells) were seeded on µ-Dish 35 mm low (80136, i-bidi) and incubated overnight. On the next day, the cells were treated with or without 3 μM MK4 1 h before the addition of 0.5 μM RSL3. Live-cell imaging was performed using 3D Cell Explorer and Eve software v1.8.2 (Nanolive). Images were obtained at 1 min intervals. During imaging, the cells were maintained at 37 °C and 5% CO₂ by using a temperature-controlled incubation chamber.

The accumulation of fatty acids was detected by staining Huh-7 cells with Oil Red O (catalog #154-02072) from Wako (Richmond, VA, USA). Huh-7 cells were cultured on coverslips in 12-well plates and treated as described above. Cells were washed with phosphate buffered saline (PBS) and fixed in 10% formalin. After washing with PBS, Oil Red O was added. Coverslips containing the cells were transferred onto the slides and mounted using aqueous mounting media. Cells were observed under brightfield (EVOS FL Cell Imaging System, Thermo Fisher Scientific), fluorescence (using FITC and DAPI Fluo channels) and phase contrast (3D Cell Explorer, NanoLive) microscopy and photographs were taken.

Escherichia coli K-12 MG1655 (E. coli) or Enterococcus faecalis NCTC 775 (E. faecalis) were incubated for 7 h at 37°C, 5% CO₂ with Luria-Bertani (LB) broth containing E171 at indicated concentrations and then fixed in 3% formalin overnight. Cells were resuspended in PBS and visualized using a Nanolive 3D cell explorer. False colors were applied to images based on refractive index using STEVE software.

MCF-7 cells were seeded in a 35-mm glass-bottom dish for 24 h and then treated with PPP at a concentration of 20 µg/mL for 3 h. The live morphology of MCF-7 cells was detected using a 3D cell holographic tomography microscope (Nanolive 3D Cell Explorer, Tolochenaz, Switzerland) and analyzed by STEVE software. During imaging, cultivation environment was maintained with sufficient amount of CO₂ at 37 °C.

For the preparation of samples, BEAS-2B cells or A549 cells were seeded into 35-mm culture dishes (IBIDI, Gräfelfing, Germany) at a density 20,000 cells/dish and incubated for 24 h. Then, cells were treated with HNTs at doses 5 and 25 µg/mL and incubated for 24 or 72 h. The dishes containing untreated cells (control) or exposed cells were placed on the tomographic microscope (3D Cell Explorer, Nanolive S.A., Lausanne, Switzerland) and three-dimensional tomographic images (z-stacks) were created. Further, post-processing steps such as background reduction and contrast enhancement were applied to the final figures using STEVE software (Nanolive).