Axio observer 3

The acridine orange/ethidium bromide dual staining assay was used to detect apoptosis. Acridine orange, a membrane-permeable dye that can stain cell nuclei, is capable of diffusing through live or dead cell membranes. On the other hand, ethidium bromide can only pass through pores of dead cell membranes and stain the cell nucleus. When acridine orange and ethidium bromide are used at the same time, fragmented bright red-orange cell nuclei will indicate (dead) late apoptotic cells, whereas live cells are only stained by acridine orange and seen as bright-green cells. This experiment was performed as indicated previously (Pajaniradje et al., 2014; Agus et al., 2018). Briefly, cells were mixed with 5 µL of acridine orange/ethidium bromide solution (60 µg/mL of AO:100 µg/mL of EB, dissolved in PBS). After incubation, cells were washed with PBS and examined under a fluorescence microscope (Carl-Zeiss, Axio Observer 3) using 40× objectives at λex = 500 nm and λem = 530 nm for acridine orange and λex = 510 nm and λem = 595 nm for ethidium bromide.

The MMP changes, MDA level and SOD activity after various treatments were measured using corresponding kits. After treatment, the MMP changes of cells were examined with mitochondrial membrane potential kit (C2006, Beyotime, Shanghai, China). Briefly, the cells were washed two times with PBS and treated with JC‐1 working solution, which was made by combining JC‐1 staining fluid (1 ml) with DMEM (1 ml) at 37°C for 20 min in the dark. The cells were then washed two times with JC-1 washing buffer. Fluorescence microscopy was used to view the images (Axio Observer 3; Carl Zeiss). The MDA level was determined using the thiobarbituric acid method and MDA detection kit (S0131S, Beyotime, Shanghai, China). SOD activity was detected using the hydroxylamine method and total SOD detection kit (S0086, Beyotime, Shanghai, China).

Blood samples were collected from the abdominal aorta and anticoagulated by heparin sodium after fasting 12 h and the last administration. Plasma was obtained from each sample by centrifuging at 3000 rpm for 10 min at 4°C. The plasma levels of TC and TG were measured according to the Kits instruction.
Part of liver tissues were fixed in 10% formalin, dehydrated, and embedded in paraffin for hematoxylin and eosin (H&E) staining. The tissues were cut into 5 µM sections by microtome (RM2245, Leica, United States) and subsequently stained with H&E. The other part of liver tissues was applied for Oil Red O staining. The frozen liver tissues were cut into 6 μM thick sections using a microtome-cryostat (NX70, Thermo Fisher Scientific, United States), air-dried on glass slides, and then fixed with 10% formaldehyde solution for 10 min. Subsequently, the sections were rinsed with distilled water and soaked with 60% isopropanol. After that, sections were performed for Oil Red O staining and hematoxylin counterstaining. Both H&E and Oil Red O stained sections were captured with a microscope (Axio Observer 3, Zeiss, Germany).

Cells were harvested for Oil Red O Staining and cellular TAG content determination according to previous studies [44 (link)]. Intracellular Oil Red O stain was extracted using isopropanol and quantified by measuring the optical absorbance at 510 nm. For staining, the cells were first washed three times with PBS, fixed in 4% paraformaldehyde for 1 h at 25 °C, washed again with PBS, and finally stained with 2 μg/mL BODIPY 493/503 (D3922; Thermo Fisher Scientific, Waltham, MA, USA) or Oil Red O for 30 min at 25 °C. The cells were counterstained with DAPI after washing with PBS and photographed using an Axio Observer 3 (Zeiss, Oberkochen, Germany). Images of the control and treated cells were captured using default parameters.

Phytophthora zoospores were induced and harvested into 1.5 mL tubes. Tubes containing 100 zoospores/μL in a 500 μL suspension were vortexed for 90 s to induce cyst formation. 500 μL cyst suspension were mixed with 500 μL 10% V8 medium and 500 μL bacterial culture (diluted to an OD600 = 1.0), and then incubated at 25°C to observe the germination of P. sojae or P. capsici. 500 μL P. infestans cyst suspension were mixed with 500 μL PEA medium and 500 μL bacterial culture (diluted to an OD600 = 1.0), and then incubated at 18°C to observe the germination, which was observed with a microscope (Zeiss Axio Observer 3). GFP-labeled P. sojae cysts were stained with 1 μg/mL chitin-binding CFW (Caleofluor White) for 5 min to make cysts clearer to observe.

For cell proliferation analyses, melb-a cells were measured by a Cell Counting Kit-8 detection kit (CCK-8; Beyotime Biotechnology). All groups of cells were seeded at a concentration of 5×10³ cells per well in 96-well plates. All experiments were carried out in triplicate at 12, 24, 48, 72, 96 and 120 h after seeding. CCK-8 solution was applied at 10 μl per well, followed by 4 h incubation at 37°C. Absorbance values of all wells were then determined at 450 nm in Multimode Microplate Reader SpectraMax M5 (Molecular Devices).
The analysis of melb-a cell migration was analyzed in a transwell migration assay. After being starved for 12 h, 2×10⁵ cells per well were seeded and cultured in the upper chamber with 200 μl RPMI-1640 medium in a 24-well plate (Corning, 3422; 8 μm pore). The bottom well contained 600 μl complete culture medium. After 24 h, the upper chamber was fixed with 4% PFA for 20 min at room temperature and then incubated with Crystal Violet (Sangon Biotech, Shanghai, China) for 20 min at room temperature. The cells in the upper chamber were then wiped off with a cotton swab, and the chamber was soaked in PBS. The cultures were photographed using a microscope (Zeiss, Axio Observer 3) to reveal the cells that had migrated to the bottom chamber. Each experiment was repeated at least three times.