The cell culture supernatant of primary hepatocytes was collected for further analysis. Triglyceride (TG) and total cholesterol (T-CHO) were measured using a triglyceride assay kit and a total cholesterol assay kit, respectively (Nanjing Jiancheng Bioengineering Institute, China). The IL-1β concentration was measured using an IL-1β ELISA kit (Lianke Biotechnology Company, China). Plasma from mice was centrifuged, separated and stored at -80 °C for further analysis. Plasma levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were measured using MDA, SOD and GSH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). The plasma level of reactive oxygen species (ROS) was measured using an ROS ELISA kit (Nanjing JiaBeiSen Biotechnology, China). ROS generation in the cell culture supernatant was assayed using a DCFH-DA fluorescent probe kit (Beyotime Biotechnology, China). Total protein was extracted from mouse liver tissues. Caspase-1 activity in liver tissues was assessed with a Caspase-1 activity assay kit (BioVision, Milpitas, CA, United States). All procedures were performed according to the manufacturers’ instructions.
Dcfh da fluorescent probe kit
Manufactured by Beyotime
Sourced in China
The DCFH-DA fluorescent probe kit is a laboratory reagent used for the detection and quantification of reactive oxygen species (ROS) in biological samples. The kit contains the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), which can be oxidized by ROS to produce a fluorescent product, 2',7'-dichlorofluorescein (DCF). This allows for the measurement of ROS levels in cells or tissues using fluorescence-based techniques.
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3 protocols using dcfh da fluorescent probe kit
1
Evaluation of Hepatocyte Oxidative Stress
2
ROS Detection using DCFH-DA Fluorescence
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Production of reactive oxygen species (ROS) was detected using DCFH-DA fluorescent probe kit (Beyotime, Shanghai, China). In brief, the plates were incubated in the dark for 20 min with DCFH-DA (10 M), and then cells were washed 3 times to remove DCFH-DA. Subsequently, cells were visualized using Leica DMIRE2 fluorescence microscope. We used 488nm excitation wavelength and 525 nm emission wavelength.
3
Intracellular ROS Detection Using DCFH-DA
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The 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe kit (Beyotime) was used to detect the intracellular ROS concentration according to Zou's method (15) (link). After the cells were washed 3 times with sterile PBS buffer, the DCFH-DA probe was diluted with serum-free RPMI 1640 medium at 1:1000. The cells were re-suspended in the diluted DCFH-DA probe suspension and incubated for 20 min at 37 °C. The cell suspension was mixed every 5 minutes so that the probe was in full contact with the cells. Finally, the cells were washed with a serum-free cell culture medium 3 times to remove the DCFH-DA probe that did not bind to the cells. The cells were re-suspended with sterile PBS buffer according to the kit instructions. The intracellular ROS expression was detected by Flow Cytometer (BD Biosciences, USA).
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