Dcfda cellular reactive oxygen species detection assay kit

U937 cells were seeded onto 96-well microplates at a final concentration of 1 × 10⁵ cells per well and treated with mesonol A or B (5 or 10 µM) for 30 min with or without pretreatment with N-acetylcysteine (NAC) (10 mM) for 1 h. The cells were stained with 2′,7′-dichlorofluorescin diacetate (DCFDA) Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, Cambridge, UK) solution (20 µM) and then incubated at 37 °C for 30 min in the dark. Fluorescence measurements were performed with a fluorescence plate reader (Bio-Rad, Hercules, CA, USA) at Ex/Em = 485/535 nm in end point mode in the presence of compounds, medium, or buffer. All experiments were performed in triplicate.

ROS generation in primary hepatocytes was measured using 2′,7′–dichlorofluorescin diacetate (DCFDA) Cellular Reactive Oxygen Species Detection Assay Kit (Abcam) as previously (44 (link)). Briefly, primary hepatocyte from WT or Rev-Erbα−/− mice livers were isolated and seeded to the black wall, clear-bottom 96-well microplate overnight before the indicated treatments; DCFDA solution was added and incubated indicated time. The fluorescence was measured with excitation at 485 nm and emission at 535 nm using a microplate reader (Bio-Tek).

The DCFDA Cellular Reactive Oxygen Species Detection Assay Kit (ab113851; Abcam) was used to estimate the reactive oxygen species (ROS) levels in cells [57 (link)]. Briefly, 2.5 x 10⁴ cells per well were seeded in dark, clear bottom 96-well microplates and allowed to adhere overnight. Then, the cells were incubated with 25 μM DCFDA for 1 h at 37°C. The fluorescence was analyzed using a fluorescent plate reader at an excitation wavelength at 485 nm and an emission wavelength at 535 nm [58 (link)].

Cellular ROS was measured in Hep3B or HepG2 cells treated with PB (120 and 240 μg/mL), corresponding polyphenols, or water control for 5 h, with or without 1-h pretreatment with 5 mM NAC, by using a 2′-7′-dichlorofluorescin diacetate (DCFDA)-Cellular Reactive Oxygen Species Detection Assay Kit (ab113851, Abcam) according to the manufacturer’s protocol. Cells were analysed on a fluorescent plate reader (Berthold Technologies, TN, USA), and mean ± standard deviation was plotted for three replicates from each condition. Tert-butyl hydrogen peroxide (TBHP), which mimics ROS activity to oxidize DCFDA to fluorescent DCF, was used as positive control.

The membrane permeable fluorescence dye JC-1 (no. T3168; Thermo Fisher Scientific) is a mitochondria membrane potential (MMP) probe that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (525 nm) to red (590 nm). This depolarization of MMP occurs at early stages of oxidative stress and cell death. This potential-sensitive emission shift from green to red is due to concentration-dependent formation of red fluorescent J aggregates, which in turn is dependent on MMP. Thus, decreases in MMP are measured by decreases in the intensity of emitted red fluorescence. Isolated mitochondria (10 ul/well) in HBSS (without Ca²⁺/Mg²⁺, phenol red no.14185-052; Thermo Fisher Scientific) were incubated with JC-1 (5.0 ug/ml in NADH-containing Complex I assay buffer; MS141, Abcam) for 1–2 min and then treated with antimycin. JC-1 emission at 525/595 nm was recorded (2 readings/min for 30 min) using a fluorescence spectrophotometer (Flex Station 3; Molecular Devices). The rate between two time points (emission at 595 nm/min) was calculated in the most linear range of decline for JC-1 emission. ROS levels were determined employing the 2’,7’-dichlorofluorescin diacetate (DCFDA) Cellular Reactive Oxygen Species Detection Assay Kit (ab113851; Abcam) according to the manufacturer’s instructions.