Eclipse ti2 u

For ROS detection, human astrocytes were seeded into 12-well plates (Falcon, Glendale, CA, USA) and transfected with specific siRNAs. After approximately 16 h, the cells were treated with water or 0.5 mg/mL KRGE for 23.5 h in serum-free DMEM media. Then, the astrocytes were treated with 20 μM H₂DCFDA (Thermo Fisher Scientific) for 30 min at 23 ± 2 °C. Finally, the cells were washed gently with PBS for 10 min. Images were captured using an inverted microscope (Eclipse Ti2-U; Nikon).

The HGC-27 and AGS cells at 48 h post transfection were seeded into 24-well plates (2×10⁴) and cultured with 5-ethynyl-2′-deoxyuridine (EdU; 50 µM; Guangzhou RiboBio Co., Ltd.) at 37°C for 2 h. The cells were fixed in 4% paraformaldehyde at 25°C for 30 min and incubated with glycine at 37°C for 4 h. After being submerged into the Apollo reaction cocktail (deionized water 469 µl, Apollo reaction buffer 25 µl, Apollo catalytic solution 5 µl, Apollo 567 fluorescent dye solution 1.5 µl and Apollo buffer additive 5 mg) at 37°C for 30 min in the dark, the cells were washed with 0.5% Triton X-100. Cell nuclei were stained using Hoechst 33342 at 37°C for 30 min. Cells were then imaged using a Nikon fluorescence microscope (Eclipse Ti2-U; Nikon Corporation; magnification, ×400).

Murine tracheal tissues were dissected, added to high-glucose DMEM containing 10% FBS
(Gibco, Grand Island, NY,USA), opened on the dorsal side and minced under stereoscopic
magnification to yield ~5 mm tissue fragments. These tissues were then transferred to a
confocal dish (BDD012035, BIOFIL, Guangzhou, China) and a scotch tape spacer was used to
facilitate their imaging under a 40× objective (CFI S Plan Flour ELWD NAMC) with an
inverted microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan).

Intracellular mitochondria activity was obtained from fluorescence imaging using 1 μM of Mitotracker (Thermo Fisher Scientific, Waltham, MA, USA), a mitochondrial membrane potential-sensitive dye. Astrocytes plated on 18 mm round coverslips in 12-well plates and were cultured to 80% confluency. The cells were subjected to distilled water or 500 μg/mL KRGE for 23.5 h, then 1 μM of Mitotracker was added for 30 min. Following washing with PBS, the cells were fixed in 4% paraformaldehyde for 10 min. They were washed with 0.1% → 0.2% → 0.1% (vol/vol) Tween 20 in PBS, each for 10 min. The blocking step, using 3% BSA prepared in PBST (i.e., PBS containing 0.1% Triton X-100), was introduced for 1 h at room temperature, followed by overnight incubation with rabbit anti-Tom20 antibody (1:3000, Abcam) prepared in PBST (PBS containing 0.1% Triton X-100) at 4 °C. After washing with 0.1% → 0.2% → 0.1% (vol/vol) Tween 20 in PBS, each for 10 min, the cells were incubated with FITC-conjugated donkey anti-rabbit IgG (1:300, Jackson ImmunoResearch) for 1 h at room temperature. We consequently placed a round cover glass over each well by using a mounting agent (Fluoro-Gel II with DAPI; Electron Microscopy Sciences). The fluorescent images were obtained using a microscope (Eclipse Ti2-U; Nikon, Tokyo, Japan).

Cells were seeded in 6-well plates at a concentration of 5 × 10⁵ cells/well and cultured until they achieved a confluent monolayer. Using a sterile 10-μL pipette tip, uniform scratches were created across the monolayer. Afterward, the plates were gently rinsed with Dulbecco’s phosphate-buffered saline to remove any detached cell debris. Subsequent to this, specific groups of cells were treated with either DNX or VitC, while a control group remained untreated. Following a 2 h incubation, all groups, excluding the control, were subjected to UVA (24 J/cm²) and UVB (100 mJ/cm²) radiation. Observations of the wound healing process were systematically made using an inverted microscope (Nikon, Eclipse Ti2-U, 10× objective) at specified intervals: 0, 6, 12, and 24 h post scratching. To assess cell migration, the percentage of the remaining cell-free region in comparison to the original scratched area was calculated. This study’s findings were based on data aggregated from at least three independent experiments. For a more detailed analysis of the wound gap, the Image-Pro Plus 6.0 Software was employed. The rate of wound closure was ascertained using Equation (2).
$$\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{C}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e} (\%)=\left(\left({\mathrm{W}}_0\right.\right.-\left.{\mathrm{W}}_{\mathrm{t}}\right)/\left.{\mathrm{W}}_0\right)\times 100$$
${\mathrm{W}}_0$ and ${\mathrm{W}}_{\mathrm{t}}$ represent the wound sizes at hours 0 and t, respectively.

Primary human brain astrocytes were seeded in 12-well plates (Corning). After fixing with 4% formaldehyde for 15 min, the cells were washed with 0.1% [v/v] Tween 20 in PBS [PBST] for 10 min, 0.2% PBST for 10 min, and 0.1% PBST for 10 min and incubated with BSA (3%) in PBS containing 0.1% Triton X-100 (SigmaAldrich) for 1 h at room temperature. Samples in 12-well plates were then treated with rabbit anti-HIF-1α primary antibody (1:400, Novus Biologicals, Littleton, CO, USA) or mouse anti-SIRT3 primary antibody (1:300, Santa Cruz Biotechnology) in PBS containing 0.1% Triton X-100 overnight at 4 °C. After washing with 0.1% [v/v] Tween 20 in PBST, 0.2% PBST, and 0.1% PBST, each for 10 min, the secondary antibodies were diluted in PBS containing 0.1% Triton X-100 for 1 h at 23 ± 2 °C. Secondary antibodies (Jackson ImmunoResearch) were a mixture of TRITC (1:400)- and/or FITC (1:300)-conjugated donkey immunoglobulin G (IgG). After the final wash, the cells were mounted onto each well using Fluoro-Gel II containing DAPI mounting solution. The images were acquired using a fluorescence microscope (Eclipse Ti2-U; Nikon). In each image, the average intensity of ten randomly chosen cells was described.