Ros assay kit

H9C2 cells were fixed with 4% paraformaldehyde for 15 min and were stained with Hoechst 33258 (Biyuntian Biotechnology Co., Ltd., China) for 30 min in the dark. Next, H9C2 cells were observed under an inverted fluorescence microscope (Leica Microsystems GmbH).
The ROS assay was conducted according to the manufacturer’s instructions of the ROS Assay Kit bought from Biyuntian Biotechnology Co., Ltd. Next, 10 μM DCFH diluted in DMEM was added to each well, followed by incubation for 30 min at 37°C. The cells were then observed under an inverted fluorescence microscope (Leica Microsystems GmbH).

After preimplantation embryos were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.5% Triton-X-100 for 40 min at room temperature, ROS studies were determined using a ROS Assay Kit according to the manufacturer's instructions (Biyuntian Bio-technology Co., Shanghai, China). After three washes in PBS, preimplantation embryos were placed in DAPI for anti-quenching and nuclear staining. Signal intensities for A488 (green fluorescence) Ex/Em: 493/630 nm were detected under a CLSM.

Intracellular ROS levels was detected by the ROS assay kit (Biyuntian Biotechnology, Hang Zhou, China). BV2 cells were divided into 6 groups and seeded in different small dishes, pretreated with tested compounds (4h at 30, 15, 7.5 μM and donepezil at 15 μM) for 1 h, and then incubated with LPS for another 24 h. The control group involved culturing the BV2 microglia cells in normal culture media without any treatment. Then, the cells were washed with PBS (pH 7.2) and incubated with DCFH-DA at 37 °C for 20 min. Finally, cells were washed with DMEM and the fluorescence intensity was observed under confocal laser scanning microscope.

The intracellular ROS level was measured using a ROS Assay Kit (Biyuntian Biotechnology, Nanjing, China). The cells were plated into 6-well plates at a density of 1 × 10⁵/well. Cells in each experimental group were induced with 200 μM H₂O₂ for 2 h and then incubated in a medium with or without NMN and CoQ10 for 24 h. Cells were washed with Phosphate Buffered Saline (PBS) and stained according to the kit manufacturer’s instructions.

The experimental group treated with 5mM butyrate for 48 h: Mitochondrial ROS was qualitatively determined by a superoxide fluorescent probe (40778ES50, Yeasen Biotechnology Co, Ltd. Shanghai, China). The experimental group treated with 0.22µM rotenone for 48 h: ROS was qualitatively determined by the ROS Assay Kit (S0033S, Beyotime Biotechnology Co, Ltd. Shanghai, China). HeLa cells were cultured in 12-well plates containing climbing sheets for 24 h. The experimental group was treated with butyrate for 48 h, followed by three washes with PBS. ROS working solution, an ethidium bromide (EB) derivative that can be rapidly oxidized by ROS, was added and the plates were incubated at 37 °C in the dark for 10 min. The sealed samples were examined with the fluorescence microscope (TOKYO, CKX3-SLP).
Flow cytometry was also employed for qualitative detection of ROS. HeLa cells were seeded in 6-well plates and treated with butyrate/rotenone for 48 h. After treatment, ROS working solution was added and incubated in the dark for 10 min at 37 °C. The ROS expression was detected by flow cytometer (Becton Dickinson, FACSCantoII).