Eclipse ts100 epi fluorescence microscope

Cells (1 × 10⁵) were seeded on coverslips (SPL, Republic of Korea) in a 6-well plate and allowed to adhere overnight. Cells were treated with vitexin (24 h) and 500 nM rapamycin as autophagy stimulator for 12 h. Cells were stained in dark with 100 μg/ml of AO in PBS for 10 min and washed with PBS. Fluorescent micrographs were obtained using a fluorescence microscope at 40× magnification. (Nikon Eclipse TS100 Epi-fluorescence microscope, Nikon Corp., Tokyo, Japan).

The effect of C₁₂O₃TR on P. digitatum cell wall integrity was determined as described by OuYang et al. (2019) (link), with some modifications. P. digitatum conidia (1 × 10⁴ CFU/mL, 90 μL) mixed with 5% PDB were cultured at 25°C for 48 h. C₁₂O₃TR with the final concentrations of 6.25 μmol/L (MIC) was added and then incubated for 0, 2, and 12 h. PBS (pH 7.0) was used as control. Each sample was then stained with 50 mg/L Calcofluor White (CFW) for 5 min in dark, and the fluorescence was examined and photographed by the Eclipse TS100 epifluorescence microscope (Nikon Corporation, Japan) with DAPI filter sets. The reproducibility of experiment results was confirmed by three replicates.

Cells were seeded on coverslips (SPL, Republic of Korea) and treated with vitexin and rapamycin (500 nm). Cells were fixed and stained using the Cyto-ID^TM Autophagy Detection Kit (Enzo Life Sciences, Plymouth Meeting, PA) according to the manufacturer's instruction and counterstained with Hoechst 33342. Coverslips were mounted on glass slides with antifade (Invitrogen, USA) staining solution and analyzed using a fluorescence microscope. (Nikon Eclipse TS100 Epi-fluorescence microscope, Nikon Corp., Tokyo, Japan).

H₂DCFDA is a widely used compound for directly measuring the redox state of cells. H₂DCFDA is deacetylated by esterases and then oxidized by ROS to form the highly fluorescent product 2’,7’-dichlorofluorescein [19 (link)]. HCT-116 cells treated with various concentrations of HMF for 24 h and 100 μM of HMF for 6–24 h before staining with 20 μM H₂DCFDA for 15 min in the dark. Cells were then washed with PBS and visualized with a fluorescence microscope (Nikon Eclipse TS100 Epi-fluorescence microscope, Japan).
To examine the accumulation of mitochondrial superoxide, cells treated with various concentrations of HMF for 24 h and 100 μM of HMF for 6–24 h, were incubated with 5 μM MitoSOX Red mitochondrial superoxide indicator (Invitrogen) for 10 min, and then were washed twice with PBS. Fluorescent images were captured using a fluorescence microscope. Mean fluorescence intensity was quantified using ImageJ software after background staining correction.

The membrane permeability of mycelia was analyzed by the propidium iodide (PI) staining method [19 (link)]. Two-day-old mycelia from PDB were collected and treated with p-anisaldehyde (0, 1/2 MIC, and MIC) for 30, 60, and 120 min. The treated mycelia were dyed with 10 μg/ml PI for 5 min at 30°C, then centrifuged at 4,000 ×g for 15 min after being washed three times with PBS. The mycelia were photographed with an Eclipse TS100 epifluorescence microscope (Nikon Corporation, Japan). The fluorescence values were determinated by a F97 PRO fluorescence spectrophotometer (Lengguang Technology, China) with an excitation wavelength of 535 nm and the emission wavelength at 615 nm. The results were expressed as the fluorescence value folds.

Alterations in mitochondrial membrane potential (MMP) were determined by using the fluorescent dye Rh-123 staining method as reported earlier [21 (link), 22 (link)]. Briefly, cells were seeded in a 6-well plate and treated with different concentrations of HMF for 24 h. Cells were washed with PBS and stained with 1 μg/ml of Rh-123 at 37°C for 30 min in the dark. After washing with PBS, fluorescence intensity of cells was analyzed under a fluorescence microscope (Nikon Eclipse TS100 Epi-fluorescence microscope, Japan). Mean fluorescence intensity was quantified using ImageJ software after background staining correction.