Seeding the cells on 6-well plates (Cornig Costar, Fisher Scientific) one day prior to the experiment was performed to assure 50-70% confluence. Staining for flowcitometric analysis was performed using total ROS Detection Kit ( Enzo Life Sciences). Briefly, following treatment, samples were suctioned to remove media and detached using sterile scraping technique. Collection of cells in 5 mL polystyrene tubes and staining (30 min, 37˚C) with the ROS detection solution provided by the manufacturer was performed. Reactive oxygen species accumulation was evaluated using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) with green (FL1 channel-530 nm) The total ROS Detection Kit (Enzo Life Sciences) was used for cell cultures staining following administration of MWCNTs-PEG and Laser irradiation. Next, suspensions of Panc-1 cells were analyzed by flow cytometry in accordance with the manufacturer's instructions.
Total ros detection kit
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom
The Total ROS Detection Kit is a fluorometric assay designed to measure the total reactive oxygen species (ROS) levels in various samples. The kit includes reagents and protocols to quantify the cumulative ROS production in a simple, rapid, and sensitive manner.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Mitosox, by Enzo Life Sciences (2 mentions)
Color matched beads, by BD (2 mentions)
Tom20 antibodies, by Novus Biologicals (2 mentions)
Cd66b clone g10f5, by BioLegend (2 mentions)
Dnase, by Roche (2 mentions)
Mitosox, by BD (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Griess reagent kit, by Thermo Fisher Scientific (1 mentions)
H2dcfda solution, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
Rat il 1β, by R&D Systems (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rat insulin elisa kit, by ALPCO (1 mentions)
Apoptotic dna ladder detection kit, by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
52 protocols using total ros detection kit
1
Measurement of Cellular ROS Levels
2
Quantifying Oxidative Stress in Cell Cultures
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For the quantification of cellular oxidative stress, cell cultures were carried out in standard growth medium without phenol red for 7 days in the presence or absence of bFGF (5 or 10 ng/mL). Nitric oxide (NO) concentration was assessed with the Griess Reagent Kit, reactive oxygen species (ROS) with a H2DCF-DA solution (both purchased from Life Technologies). Antioxidant potential of ASCs was determined on the basis of superoxide dismutase (SOD) secretion levels detected by SOD Assay Kit. Additionally, total ROS in cells was detected by means of fluorescence microscopy, using Total ROS Detection Kit (Enzo Life Sciences). Furthermore, to determine whether mitochondria stand as a major source of ROS within the cells, cells were exposed to rhodamine-based dye, MitoRed, in 1 : 1000 dilution. All procedures were conducted following the protocols supplied by the manufacturers.
3
Quantifying Oxidative Stress in IEC Cells
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The Total ROS Detection Kit (Enzo Life Sciences Inc., New York, USA) was used to evaluate the levels of oxidative stress in IEC-6 and IEC6shAtg5 cells[9 (link)] before and after H2O2 treatment. Fluorescence was directly measured with a GloMax Multi-Detection System.
4
Neutrophil ROS Detection Assay
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Total ROS detection kit (Enzo) was used to evaluate the production of ROS by neutrophils, as described in the manufacturer’s protocol.
Flow cytometry acquisition was performed using a FACSCalibur (BD Biosciences) and data were analyzed using Summit v4.3 software.
Flow cytometry acquisition was performed using a FACSCalibur (BD Biosciences) and data were analyzed using Summit v4.3 software.
5
Intracellular ROS Detection Assay
6
Quantifying Cellular ROS Levels
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To determine reactive oxygen species (ROS) level, treated cells were measured using the Total ROS Detection Kit according to the manufacturer's protocol (Enzo; Catalog No. 51011). The cells (1 × 105 cells) were grown on 10-cm plates overnight and treated with vehicle (DMSO, 0.5%), DET (11 μM), DETD-35 (3 μM), or PTX (1 μM) for 1 h. Then, the cells were collected and resuspended in ROS detection solution (green probe) that directly reacts with a wide range of reactive species, for example hydroxyl radicals, hydrogen peroxide, peroxynitrite, peroxy radical, and nitric oxide, and the cells were further stained for 30 min at 37°C. After staining cells, the ROS level of treated cells was determined by flow cytometry.
7
Intracellular ROS Detection by Flow Cytometry
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Intracellular ROS levels were measured using a Total ROS Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) in accordance with the manufacturer’s instructions. ROS fluorescence was detected using a FACSVerse flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo version 10.0 software (Becton-Dickinson, Franklin Lakes, NJ, USA). The mean fluorescence of ROS production was determined automatically and presented as the geometric mean.
8
Mitochondrial ROS and Membrane Potential
9
Assessing Mitochondrial Function in Neutrophils
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The total ROS detection kit and MitoSOX (2.5 µM) were used according to the manufacturer’s instructions (Enzo Life Sciences and Life Technologies, NY). Lupus LDGs and control NDGs were incubated with either MitoSOX (5 µM) or mitoTracker® green FM (100 nM) and cells were quantified by flow cytometry following manufacturer’s instructions for concentration and timing and using BD color matched beads for compensation (catalog number: 552843 BD Biosciences, San Diego, CA). Mitochondria were also analyzed by flow cytometry using TOM20 antibodies (Novus Biologicals) and MitoSOX (Life Technologies). In some experiments, neutrophils were treated with pronase (2 mg/mL, Calbiochem, San Diego, CA) or DNase (50 µg/mL, Roche) 30 minutes prior to addition of TOM20. Neutrophil activation was assessed by cell surface expression of CD66b (clone G10F5, BioLegend). Phagocytosis of 8-OHdG ICs was analyzed by flow cytometry and immunofluorescence microscopy upon incubation of neutrophils with Alexa Fluor-conjugated 8-OHdG ICs (2.5 µg/mL, pre-formed as described above) for 30 minutes. Phagocytosis index was determined as phagocytosed ICs per neutrophil and field of observation. Data were analyzed by FlowJo (Tree Star Inc, Ashland, OR). For all analyses, isotype antibodies or fluorescently labeled beads were used as negative controls.
10
Liposomal Formulations on NO Release
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To evaluate the effect of liposomal formulations on the release of NO, differentiated HL60 cells were seeded at 100,000 cells per well in a 96-well plate in fresh medium supplemented with the respective treatments of EPA-liposomes or free EPA at the indicated concentrations in the presence of 100 ng/mL LPS. After 24 h of incubation, the cell-culture medium was removed pretreatment or in the pre-stimulation setup, and the supernatant was collected for a nitric oxide assay with Griess reagents (EnzoLife Sciences, Farmingdale, NY, USA) following the attached protocols. The NO levels were represented by the absorbance was measured at 550 nm on a plate reader. The collected cells were resuspended in a white 96-well plate for ROS measurement using a total ROS detection kit (Enzo Life Sciences, Farmingdale, NY, USA) per the guidance of the manual.
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