We used two methods to measure the levels of superoxide and ROS in HUVECs. In the first method, cells were transfected with NCF1 splicing isoforms for 48 hours, then incubated with hydrocyanine 3 (H-cy3) (provided by Dr. Niren Murthy’s lab) for 15 minutes. H-cy3 is a fluorescent sensor that can react with ROS in live cells, which enables a direct observation and measurement under a fluorescent microscope [12 (link)]. Then the cells were washed and challenged with phorbol myristate acetate (PMA) 100 nM for 1.5 hours. The fluorescent intensity was quantified with the Image-J software, which indicated the superoxide level. In the second method, cells were transfected with NCF1 splicing isoforms for 48 hours, then HUVECs were washed and incubated with ROS/superoxide detection mix (Enzo Life Sciences, Farmingdale, NY, USA, Total ROS/Superoxide Detection Kit) for 1 hour, and treated with pyocyanin (Enzo Life Sciences, Total ROS/Superoxide Detection Kit) 50 μM in growth medium for 1 hour. The fluorescent signals were imaged with fluorescent microscope and quantified with the Image-J software. This method measures the levels of superoxide and general ROS simultaneously.
Total ros superoxide detection kit
Manufactured by Enzo Life Sciences
Sourced in United States, Germany, United Kingdom
The Total ROS/Superoxide Detection Kit is a lab equipment product designed to measure the total levels of reactive oxygen species (ROS) and superoxide in biological samples. The kit provides reagents and protocols for a colorimetric or fluorometric assay to quantify these species.
Automatically generated - may contain errors
Lab products found in correlation
Facscan flow cytometer, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Nucleocounter nc 3000 system, by ChemoMetec (3 mentions)
Ros id total ros superoxide detection kit, by Enzo Life Sciences (2 mentions)
Enz 51010, by Enzo Life Sciences (2 mentions)
Facsaria 2, by BD (2 mentions)
Cellquest pro software, by BD (2 mentions)
Ros glo assay, by Promega (1 mentions)
Gsh gssg glo assay, by Promega (1 mentions)
Biozero bz 8000, by Keyence (1 mentions)
Facscalibur, by BD (1 mentions)
Microplate reader, by Tecan (1 mentions)
Facsdiva, by BD (1 mentions)
Ix71 inverted fluorescence microscope, by Olympus (1 mentions)
Facscalibur flow cytometer system, by BD (1 mentions)
Trifluoroacetic acid, by Thermo Fisher Scientific (1 mentions)
Anhydrous sodium acetate, by Thermo Fisher Scientific (1 mentions)
Disodium hydrogenphosphate dodecahydrate, by Thermo Fisher Scientific (1 mentions)
Celite, by Merck Group (1 mentions)
Benzo h quinoline, by Merck Group (1 mentions)
60 protocols using total ros superoxide detection kit
1
Measurement of Superoxide and ROS in HUVECs
2
Measurement of Reactive Oxygen Species in Endothelial Cells
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After incubation with EBM-2 containing 5% FBS for 16 h, cells (3 × 103 cells/well) were seeded into 96-well dishes in EBM-2 containing 5% FBS with 25 μM of pyocyanin for 30 min. The Total ROS/Superoxide detection kit (Enzo Life Science, Plymouth Meeting, PA) and Varioskan Flash (Thermo Fisher Scientific) were used to measure ROS levels. After incubation of ECs with EBM-2 supplemented with 5% FBS (control) or 0.5% FBS (serum starvation) for 24 h, the Total ROS/Superoxide detection kit (Enzo Life Science) and FACS Aria II (BD Biosciences, San Jose, CA, USA) were used to measure the ROS levels. After incubation of ECs in normoxia (20% O2) or hypoxia (1% O2) for 24 h, ROS levels were measured with the Total ROS/Superoxide detection kit.
3
Evaluating THSG's Effects on TGF-β1-Induced ROS
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MRC-5 cells were seeded in 96-well black wall/clear bottom plates at a density of 2×104 cells per well for overnight adhesion. Before experiments, cells were serum starved for 24 h, followed by treatment with THSG (50 and 100 μg/ml) for further incubation for 24 h. The cells were subsequently incubated with 2.5 ng/ml of TGF-β1 for an additional 24 h. ROS production in cells was measured using the Total ROS/Superoxide detection kit (Enzo Life Sciences, Farmingdale, NY, United States) following the manufacturer’s instructions.
4
Quantifying Cellular Oxidative Stress
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ROS were measured using the total ROS/Superoxide Detection Kit (Enzo Life Science, Farmingdale, NY, USA) according to the manufacturer's instructions. Using a combination of two specific fluorescent probes, the kit allows for the real-time observation of global ROS levels, specifically those of superoxide, in living cells. Cells were stained with the two-color ROS Detection Kit and analyzed using the NucleoCounter NC-3000 system (ChemoMetec, Allerod, Denmark). Briefly, 2.4×105 cells were seeded in six-well plates overnight and then treated with 80 μg/ml PG or vehicle. ROS and oxidative stress of the harvested cells were detected by staining with the two fluorescent dyes from the ROS-ID total ROS/Superoxide Detection Kit (ENZ-51010; Enzo). In addition, Hoechst 33342 was used to stain the harvested cells to detect the total cell population [27 (link)].
5
Measuring Intracellular ROS and Superoxide
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Intracellular ROS and superoxide levels were measured using Total ROS/Superoxide Detection Kit (Enzo Life Sciences, Lörrach, Germany). Briefly, cells were cultured and treated with appropriate concentration of EF24 in the absence and presence of NAC. After 8 hours, media was removed and the cells were washed twice with 1x wash buffer. The cells were then trypsinized, centrifuged for 5 min at 400 x g, and further resuspended in 500 μl of ROS/superoxide detection solution. Cells were stained at 37°C for 30 min in the dark and immediately analyzed by flow cytometry.
6
Nanoparticle-Induced DNA Damage and Oxidative Stress
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Nanoparticles can cause DNA damage to human cells [22 (link)]. DNA damage in cells was assessed using an assay for γH2AX histone phosphorylation (Apoptosis, DNA Damage and Cell Proliferation Kit®, BD Biosciences). Nanoparticles can cause oxidative damage to cells that induces apoptosis [23 (link)]. Oxidative responses in VEC were measured with a Molecular Probes CellROX Green® assay (Invitrogen) that detects an oxidation-sensitive dye bound to DNA in the cells by flow cytometry and also the ROS-Glo™ assay (Promega) for H2O2 measurements by high content analysis. Superoxide production was measured (Total ROS/Superoxide Detection kit®, Enzo) and the amount of oxidized glutathione was also measured (GSH/GSSG-Glo Assay™, Promega), as a measure of oxidative damage responses in the cells.
7
Quantifying Superoxide Levels in Media-Exposed Cells
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After 3 days of exposure to four types of media (control group, control + AGEs group, DD group, DD + AGEs group), superoxide detection reagent was detected by using a Total ROS/Superoxide detection kit (Enzo Life Science, Plymouth Meeting, PA, USA), and the cells were counterstained with DAPI. Digital images were captured from five non-overlapping microscopic fields per well by using the Bio Zero BZ-8000 (Keyence, Osaka, Japan), and superoxide-positive cells were counted.
8
Quantifying ROS and Superoxide in Cells Exposed to CNTs
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Total ROS/superoxide production in cells exposed to CNTs was determined using a total ROS/superoxide detection kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions. After the cells had adhered for 24 hours in 12-well plates, they were pretreated with oxidative stress detection reagent and superoxide detection reagent for 30 minutes before CNT solution (1 μg/mL in BEAS-2B and 10 μg/mL in MESO-1 cells) was added. Pyocyanin (100 μM) was used to induce ROS production. After 60 minutes, the cells were washed once with 1× wash buffer and harvested with trypsin–ethylenediaminetetraacetic acid. Cells were resuspended in 0.3 mL 1× wash buffer with 10% FBS and passed through a nylon mesh; then, they were subjected to flow cytometry (FACSCalibur™; BD Biosciences, San Jose, CA, USA) using the FL1 and FL2 channels for oxidative stress detection reagent and superoxide detection reagent signals, respectively, until 10,000 cells were recorded. Cell suspensions were assayed in triplicate for each treatment condition.
9
Quantifying Intracellular ROS in Tenocytes
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According to previous reports [18 (link), 19 (link)], intracellular ROS levels in tenocytes of each group were detected by the oxidation-sensitive fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) using the Total ROS/Superoxide Detection Kit (Enzo Life Science, Farmingdale, NY, USA) following the manufacturer’s protocol. Tenocytes (5 × 104) were incubated with DCFH-DA at a final concentration of 10 µM for 60 min at 37 °C in the dark, washed three times with PBS, trypsinized, and resuspended. For quantification, the number of ROS-positive cells and DAPI-positive cells in four rectangular areas (0.75 mm × 1.0 mm) in each slide were counted, and the mean values were calculated. Each area was randomly selected and the cells were manually counted by two blinded investigators. The percentage of ROS-positive cells was calculated using the formula (number of ROS-positive nuclei/number of DAPI-positive nuclei) × 100 and expressed as the mean of the four areas (n = 15 per group).
10
ROS/Superoxide Analysis of A2780 Cells
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Flow cytometry analysis of ROS/superoxide
generation in A2780 cells caused by exposure to complex2 was carried out using the Total ROS/Superoxide detection kit (Enzo-Life
Sciences) according to the instructions. A total of 1.5 × 106 A2780 cells per well were seeded in a six-well plate. Cells
were preincubated in drug-free media at 310 K for 24 h in a 5% CO2 humidified atmosphere, and then drugs were added to triplicates
at IC50 concentration. After 24 h of drug exposure, supernatants
were removed by suction and cells were washed and harvested. Staining
was achieved by resuspending the cell pellets in buffer containing
the orange/green fluorescent reagents. Cells were analyzed in a Becton
Dickinson FACScan flow cytometer using FL1 channel Ex/Em 490/525 nm
for the oxidative stress and FL2 channel Ex/Em 550/620 nm for superoxide
detection. Positive controls were obtained by exposure of cells to
pyocyanin for 30 min. Data were processed using Flowjo software. At
all times, samples were kept under dark conditions to avoid light-induced
ROS production. Welch’s t-tests were carried
out to establish statistical significance of the variations.
generation in A2780 cells caused by exposure to complex
Sciences) according to the instructions. A total of 1.5 × 106 A2780 cells per well were seeded in a six-well plate. Cells
were preincubated in drug-free media at 310 K for 24 h in a 5% CO2 humidified atmosphere, and then drugs were added to triplicates
at IC50 concentration. After 24 h of drug exposure, supernatants
were removed by suction and cells were washed and harvested. Staining
was achieved by resuspending the cell pellets in buffer containing
the orange/green fluorescent reagents. Cells were analyzed in a Becton
Dickinson FACScan flow cytometer using FL1 channel Ex/Em 490/525 nm
for the oxidative stress and FL2 channel Ex/Em 550/620 nm for superoxide
detection. Positive controls were obtained by exposure of cells to
pyocyanin for 30 min. Data were processed using Flowjo software. At
all times, samples were kept under dark conditions to avoid light-induced
ROS production. Welch’s t-tests were carried
out to establish statistical significance of the variations.
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