All the materials used were of the highest grade of purity. No further purification was necessary. D-galactose (G5388), L-galactose (G7134), galactose 1-phosphate (G0380), galactitol (D0256), human tubular fluid (HTF) medium, M2 medium, anti-α tubulin antibody, fluorescein isothiocyanate (FITC) conjugate anti-goat antibody, 4′,6′-diamino-2-phenylindole (DAPI), 1% Bovine Serum Albumin (BSA), 0.1% M Glycine, and 0.1% Triton X- 100 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Normal Goat Serum (2%) was from Invitrogen (Grand Island, NY), 0.2% Powder Milk (Nestle) from grocery and anti-fade agent was obtained from Biomedia, CA. Cellular reactive oxygen species (ROS) detection assay kit (ab186029, Abcam, Cambridge, United Kingdom) and In Situ Cell Death Detection kit, AP (11684795910, Roche Applied Science, Penzberg, Germany) were procured from respective commercial sources.
Cellular reactive oxygen species detection assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Cellular Reactive Oxygen Species Detection Assay Kit is a fluorometric assay designed to detect and quantify reactive oxygen species (ROS) in live cells. The kit contains a cell-permeable fluorogenic probe that reacts with various ROS, resulting in a fluorescent signal that can be measured using a fluorescence microplate reader or microscope.
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Lab products found in correlation
Nis element, by Nikon (3 mentions)
Eclipse 90i epifluorescence microscope, by Nikon (3 mentions)
Fluorescence microplate reader, by PerkinElmer (2 mentions)
Synergy h1 hybrid, by Agilent Technologies (2 mentions)
Facscalibur, by BD (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Galactitol, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
D galactose g5388, by Merck Group (1 mentions)
Fluorescein isothiocyanate fitc conjugate anti goat antibody, by Merck Group (1 mentions)
M glycine, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (1 mentions)
M2 medium, by Merck Group (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Varioskan flash multimode reader, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
Mtt assay, by Merck Group (1 mentions)
60 protocols using cellular reactive oxygen species detection assay kit
1
Galactose Metabolism and Oxidative Stress
2
Oxidative Stress Evaluation in H9c2 Cells
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H9c2 cells (2.5 × 103 cells/well) were seeded into a 96-well plate. After overnight attachment, cells were stained with DCFDA for 45 min and then examined using a Cellular Reactive Oxygen Species (ROS) Detection Assay Kit (Abcam) following the manufacturer’s instructions. H9c2 cells (2.5 × 103 cells/well) were seeded into a 96-well plate. After attachment, cells were lysed using lysis buffer to obtain the protein sample. Then, samples were detected using a Human/Mouse/Rat Total superoxide dismutase (SOD) 2/Mn-SOD DuoSet IC ELISA kit (R&D Systems, Minneapolis, MN, USA). H9c2 cells (1 × 104 cells/well) were seeded into a 24-well plate. After treatment, the supernatant was collected to determine the levels of lactate dehydrogenase (LDH) and malondialdehyde (MDA) using relevant assay kits (Abcam).
3
Oxidative Stress-Induced ROS Evaluation
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ROS generation was evaluated in SIRC cells, after oxidative stress induction by treatment with H2O2, by using the 2′,7′–dichlorofluorescin diacetate (DCFDA)– Cellular Reactive Oxygen Species Detection Assay Kit (ab113851, Abcam, Cambridge, United Kingdom) according to the manufacturer’s protocol, as previously described by Maugeri et al. (Antioxidants 2022. PMID: 35052632). Briefly, SIRC cells were plated into 96-well plates (1 × 104 cells/well). After overnight growth, cells were cultured for 60 min in the control medium (CTR); or in the presence of the formulation #3, containing 0.32% sodium hyaluronate (SH-CL), 0.5% taurine, 0.05% vitamin B6 and 0.05% vitamin B12 (RenerviX®); or in the presence of formulation #6, containing 0.32% sodium hyaluronate (SH-CL) and 0.5% taurine. Then, oxidative stress was induced with 0.3 mM H2O2 treatment for 30 min. Subsequently, cells were washed gently in PBS twice and incubated with 25 μM DCFDA previously dissolved in a buffer solution for 45 min in the dark. ROS concentration was detected by fluorescence spectroscopy with excitation and emission wavelength of 495 nm and 529 nm, respectively, using Varioskan Flash Multimode Reader (Thermo Fisher Scientific). Twelve replicate wells were used for each group.
4
Cellular ROS Detection by Flow Cytometry
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For cellular ROS detection, cells were incubated in a humidified chamber at 37°C with 5% CO2 for 30 minutes with CM-H2DCFDA or Cellular Reactive Oxygen Species Detection Assay Kit (Deep Red) (Abcam, ab186029) in physiological buffer according to the manufacturer’s protocol. After incubation, cells were resuspended in tubes and examined by flow cytometry within 2 hours, and the signals from the FITC or APC channel were monitored.
For ROS levels in medium or tumor supernatants, a DCF ROS/RNS Assay Kit was used according to the manufacturer’s protocol.
For ROS levels in medium or tumor supernatants, a DCF ROS/RNS Assay Kit was used according to the manufacturer’s protocol.
5
Measuring Cellular ROS Levels
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Cells in log phase of growth were grown to subconfluency, detached and seeded into 96-well plates at a concentration of 2.5x104 cells per well. ROS activity was measured using the Cellular Reactive Oxygen Species Detection Assay Kit (Abcam) according to manufacturer protocol. Briefly, cells were allowed to attach overnight, washed then treated with or without compound at indicated concentrations and timepoints. Cells were then exposed to 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) for 40 minutes and fluorescence was measured at 485/535 nm on a Tecan microplate reader.
6
ROS Detection in K562 Cells
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The total intracellular superoxide and hydroxyl radicals in the K562 cells was detected using the Cellular Reactive Oxygen Species Detection Assay Kit (ab186027, Abcam, Cambridge, UK), following the manufacturer’s instructions. Briefly, the K562 cells were plated (5 × 104/100 µL per well) in a 96-well plate overnight. Subsequently, cells were treated with different doses of compound 3d and stained with ROS red stock solution for 2 h at room temperature. After incubation, a fluorescence microplate reader (PerkinElmer, Waltham, MA, USA) was used to detect the intensity of the fluorescence at 520 nm and 605 nm excitation and emission wavelengths, respectively.
7
Cell Viability and Oxidative Stress Assay
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Culture media and supplements were purchased from Innoprot (Elexalde Derio, Spain) or Lonza (Basel, Switzerland). T25 flasks and multiwell culture plates were purchased from Corning (New York, NY, USA). MTT assay was purchased from Chemicon (Temecula, CA, USA). Cellular Reactive Oxygen Species Detection Assay Kit was purchased from Abcam (Cambridge, UK).
8
Measuring Cellular ROS Production
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ROS production was measured in the 12 LCLs cultured in media treated with PMA-Io, seeded at 5000 cells per well in 96-well microplates for 0.5, 2, 4, and 6 h, using the Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, ab113851), following the manufacturer’s instructions. This kit uses the cell permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFDA) to measure ROS activity within the cell. The resulting oxidation of DCFDA by intracellular ROS was detected by fluorescence spectroscopy with an Infiniti®Lumi Microplate Reader (Tecan, Mannedorf, Switzerland) with maximum excitation and emission spectra of 495 and 529 nm, respectively.
9
Intracellular ROS Quantification by Microscopy
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The intracellular ROS were measured using Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, U.S.A.). The cells adhering to the coverslip were incubated with 20 µM ROS Red Stock Solution at 37°C in the dark for 30 min. Hoechst 33342 was added at a final concentration of 1 µM to the ROS Red Stock Solution staining solution during the last 5 min of the incubation. The cells were then washed in PBS and analyzed by confocal microscope.
10
Cellular Reactive Oxygen Assay
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The Cellular Reactive Oxygen Species Detection Assay Kit (AbCam) was used according to the manufacturer’s instructions. Cells were seeded and stimulated for 72 hr before the assay was carried out with 25 μM DCFDA. Samples were analyzed on a Synergy HI microplate reader (BioTek). Results were first normalized to a positive and negative control.
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