Intracellular ROS production was measured using dihydroethidine (DHE) dye (Assay Kit Reactive Oxygen Species (ab236206), Abcam, Cambridge, UK) in live cells, which is selective for superoxide anion and hydrogen peroxide. Briefly, following incubation with MVs in a 96 wells microplate, HepG2 cells (2 × 105/mL) were incubated with DHE (2.5 μM) for 30 min at 37 °C. Cells were washed, resuspended in PBS and analyzed for fluorescence intensity using a Microplate Fluorescence Reader (BioTek Instruments, Inc., model: FL600 FA, Winooski, VT, USA) at excitation and emission wavelengths of 480–520 nm and 570 nm, respectively. Antimycin A, an inhibitor of complex III of the mitochondrial electron transport chain, was included as a positive control for ROS generation. N-acetyl Cysteine was included as an anti-oxidant control. The level of ROS generation was represented as total DHE fluorescence intensity. Data were normalized using DAPI fluorescence intensity.
Reactive oxygen species assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Reactive Oxygen Species Assay Kit is a fluorometric method for the detection and quantification of reactive oxygen species (ROS) in biological samples. The kit includes a fluorogenic probe that reacts with various ROS, producing a fluorescent signal that can be measured using a fluorescence plate reader or other compatible detection equipment.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microplate reader, by Agilent Technologies (1 mentions)
Superoxide dismutase activity assay kit, by Abcam (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
9 protocols using reactive oxygen species assay kit
1
Measurement of Intracellular ROS in HepG2 Cells
2
Quantification of Cellular ROS Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular ROS levels were determined by MitoSOX™ Red mitochondrial superoxide indicator (Abmole, Cat#M19992), and the fluorescent values were quantified and normalized to the cell number. For in vivo ROS detection, the cell suspension was prepared from digested aorta tissues. A DHE (Dihydroethidium) Assay Kit/Reactive Oxygen Species (Abcam, Cat#ab236206) was used to measure the ROS level in live cells. Briefly, the resuspended cells were normalized and added to a V-bottom plate, and then, ROS staining buffer and Cell-Based Assay Buffer were added. The fluorescence intensity was measured using a 480 nm excitation wavelength and 570 nm emission wavelength with a microplate reader. The ROS production is expressed as total DHE fluorescence.
3
Quantifying Cellular ROS Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular ROS level was also detected by DHE fluorescence by using DHE (Dihydroethidium) Assay Kit–Reactive Oxygen Species (Abcam, Cambridge, UK) following the manufacturer’s protocol. Indicated groups of C2C12 cells seeded in 96-well plates were incubated with 5 μM DHE at 37 °C for 30 min, and the fluorescence was measured by using a microplate reader at 495 nm excitation wavelength and 580 nm emission wavelength. All procedures were protected from light.
4
Quantitative Assessment of Cellular ROS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cellular reactive oxygen species (ROS) were quantitatively assessed using the Reactive Oxygen Species Assay Kit (Abcam #ab113851) that uses the cell permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFD) following the manufacturer’s instructions (Abcam, Waltham, MA, USA). Briefly, cells were incubated under normoxic or hypoxic conditions for 48 h, then incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) probe (20 μM, 100 μL/well) for 45 min at 37 °C in the dark. The non-fluorescent DCFDA is de-acetylated by cellular esterase and then oxidized by ROS into the fluorescent 2′,7′-dichlorofluorescein (DCF), which was detected at Ex/Em 495/529 nm using the Synergy HT multi-detection microplate reader (BioTek).
5
Quantifying Antioxidant and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to manufacturer protocol, the levels of SOD/ROS in PC-12 cells or brain tissues of rats were measured using Superoxide Dismutase Activity Assay Kit or Reactive Oxygen Species Assay Kit (Abcam, Cambridge, MA, USA), respectively. SOS activity (OD450) and ROS fluorescence (OD520) were measured using a Synergy HT microplate reader (Biotek, Winooski, VT, USA).
6
Quantifying Intracellular ROS and Mitochondrial Superoxide
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment for 24 h, the intracellular ROS level was measured using a Reactive Oxygen Species Assay Kit (Abcam, Cambridge, UK), according to the kit’s instructions. The images were captured by a fluorescence microscope (Olympus) at an excitation of 484 nm and an emission of 530 nm. In addition, the MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Carlsbad, CA, USA) was employed to measure mitochondria in live cells, as recommended by the manufacturer. The images were obtained under a fluorescence microscope (Olympus) at an excitation of 488 nm and an emission of 583 nm. The fluorescence intensity was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA) in at least five fields and normalized by the number of cells.
7
Evaluating IFN-γ and E2 Effects on Cellular ROS
8
Reactive Oxygen Species Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ROS levels were determined using the Reactive Oxygen Species Assay Kit (Abcam #ab113851) as per the manufacturer's protocol. Briefly, the iJ2 cells were cleared by quick trypsinization and then the attached HFK cells were washed twice with ice‐cold PBS. The cells were incubated with 6‐carboxy‐2, 7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) at a final concentration of 20 μM for 40 min at 37°C in a dark area. The ROS levels were measured by a fluorescence microscope (EVOS).
9
Measuring Cellular Reactive Oxygen Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactive oxygen species from cellular was determined using the conversion of non-fluorescent 5, 6-Chloromethyl-2V, 7V dichloro dihydro fluorescein diacetate (CM-H2DCFDA) to its fluorescent derivative (DCF) by Reactive Oxygen Species Assay Kit (Abcam, USA) according to the manufacturer’s recommendation. Four groups of SW620 cells, respectively, treated with free CPT-11, CPT-11-loaded DSPE-PEG2000 particles, CPT-11-loaded DSPE-PEG 2000 targeting EGFR liposome, and the control group. Then, 50 μmol/L DCFH2-DA were added for 30 min, washed with PBS, and centrifuged. Furthermore, PBS containing 1% Triton X-100 was added, and the intensity of DCF fluorescence (OD) was assessed via a microplate reader at 485 nm of excitation and 530 nm of emission.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!