Eclipse te2000 s

Fluorescence microscopy studies on the cell area were performed using a Nikon Eclipse TE2000-S (Nikon Instruments, Japan) inverse fluorescence microscope with a LH-M100C mercury lamp and GFP (G) bandpass filter (exc. 480, em. 535 nm). A 20x air objective and a 10x were used. Staining of the cell body was done with Calcein AM (1:100) (Sigma Aldrich, USA). Staining of the nucleus was performed using Hoechst dye (Sigma Aldrich, USA). Micrographs were processed with the Fiji distribution of ImageJ and the ZEN Imaging Software (Zeiss). The cell area of at least three independent samples was measured for all cells in the area of the micrograph at 20 °C.

In our experiment, the changes of MMP were analyzed by 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Calbiochem, USA) staining. Cells were seeded into 6-well plates at 1 × 10⁵ cells/well and incubated with 2.5 and 5 mg/kg of TM for 6 h, following with another 15 min exposure to 2 μM of JC-1 at 37°C in darkness. After three washes, fluorescent color changes were analyzed using fluorescent microscopy under Nikon Eclipse TE 2000-S fluorescence microscope (20x; CCD camera; Nikon Instruments Inc., Japan).

Adherent epithelial cells and adherent residual cells were analyzed by immunofluorescence with anti-Mucin8-FITC or fibroblast marker-FITC (Santa Cruz Biotechnology, TX, USA) and anti-HLA-G 87G-PE (Exbio). All samples were observed under a UV light microscope (Nikon Eclipse TE2000S, Nikon, Italy).

The effectiveness of the HPH treatment in disrupting plant cells was examined by conducting microscopic analysis on both the untreated and HPH-treated suspensions. Microscopic examination was performed using an optical inverted microscope (Nikon Eclipse TE 2000S, Nikon Instruments Europe B.V., Amsterdam, The Netherlands). The microscope was equipped with a polarization filter and utilized a 10× objective lens, coupled to a DS Camera Control Unit (DS–5M–L1, Nikon Instruments Europe B.V.), for image acquisition.

MG63 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells / well. The cells were then treated with 1, 2.5, or 5 μM AU for 2 h followed by 4 μM Dex or 200 μM of H₂O₂ for 24 h. Following the incubation, the cells were harvested, resuspended in solution at a concentration of 1×10⁶ cells / mL, and stained with propidium iodide and/or Annexin V for 20 min at room temperature. The rate of apoptosis was measured using a Muse Cell Analyzer (Millipore, USA).
The MMP was evaluated by staining the cells with 2 μmoL / L 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, USA) at 37°C for 15 min in the dark. The cells were then washed three times with phosphate buffered saline (PBS) and red and green fluorescence recorded using a fluorescence microscope (20×; CCD camera, TE2000, Nikon, Japan).
Intracellular ROS was quantified by staining the cells with 10 μM 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich) for 15 min at 37°C in the dark. The cells were then washed three times with PBS and green fluorescence, which reflects the intracellular ROS level, recorded using a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).