ROS was quantitated using CM-H2DCFDA reagent from Invitrogen™ (Cat # C6827). CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for ROS generation in cells and a general oxidative stress indicator. CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols and subsequent oxidation yields a green fluorescent product that can be quantitated using fluorescent microscopy imaging (Ex/Em: ~ 492–495/517–527 nm). Microglial cells were plated on glass-bottom petri dishes and cells grow to 80% confluency. Cell were then treated with 0.5 µg/ml of SARS-COV2 spike protein for 24 h following which cells were washed with 1X PBS, following which 5 μM of CM-H2DCFDA (freshly prepared in HBSS) was added and cells incubated for 30 min in dark CO2 incubator. After 30 min, cells were washed with PBS and the ROS production was quantitated immediately by measuring the green fluorescence using the EVOS® FL Cell Imaging System (Life Technologies, Grand Island, NY).
Cm h2dcfda reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
CM-H2DCFDA is a fluorogenic dye that measures hydroxyl, peroxyl, and other reactive oxygen species (ROS) activity within living cells. It is a reduced, acetylated form of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA).
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by FlowJo (2 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Gssg gsh quantification kit, by Dojindo Laboratories (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Spectramax paradigm, by Molecular Devices (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lsm710 confocal microscope, by Adobe (1 mentions)
Flexstation, by Molecular Devices (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Prism 4, by GraphPad (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
15 protocols using cm h2dcfda reagent
1
Quantification of ROS in Microglia Exposed to SARS-CoV-2 Spike Protein
2
Quantifying Cellular Oxidative Stress
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Cellular H2O2 was assayed using the CM-H2DCFDA reagent (Invitrogen) according to manufacturer’s instructions using flow cytometry (Gallios, Beckman, CA, USA) on a microplate reader (SpectraMax i3x; Molecular Devices). To determine cell-derived ROS level, accumulated ROS in the medium was quantified by OxiSelectTM Hydrogen Peroxide Assay Kit (Cell Biolabs, Inc.). For GSH/GSSG analysis, liver or cells were homogenized with 5% 5-sulfosalicylic acid dihydrate, and GSSG and GSH levels were measured using a GSSG/GSH Quantification kit (Dojindo, Japan) according to the manufacturer’s instructions.
3
Quantifying Oxidative Stress in Cardiomyocytes
4
Quantifying Cellular Oxidative Stress
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Cellular ROS level was assayed using the CM-H2DCFDA reagent (Invitrogen Life Technologies) according to manufacturer's instructions using a microplate reader. Briefly, cells seeded in a 96-well plate were stained with CM-H2DCFDA reagent (20 μM in PBS, 10 min), and washed by PBS. The cellular fluorescence was assessed by a fluorescent microplate reader (SpectraMax Paradigm, Molecular Devices, Ex 492 nm, Em 525 nm). In some setting, cells were incubated with ML171 (10 μM), GKT137831 (100 μM), diphenyleneiodonium (DPI, 50 μM), NAC (5 mM) or vehicle (Veh) for 1 h, and followed by UVB irradiation.
To assess the intracellular H2O2 level, cells were transfected with H2O2 sensitive probe pHyPer cDNA plasmid (Evorgen) with Lipofectamine 2000 (Invitrogen) according to a manufacture's instruction [23] (link), [24] (link). G418 resistant transfectants were isolated as pHyPer stably transfected cells. Cellular fluorescence was assessed by a fluorescent microplate reader (SpectraMax Paradigm, Molecular Devices, YFP: Ex 497 nm, Em 525 nm, CFP: Ex 434 nm, Em 525 nm). YFP to CFP excited (497/434) ratio was calculated as intracellular H2O2 level [23] (link), [25] (link).
To assess the intracellular H2O2 level, cells were transfected with H2O2 sensitive probe pHyPer cDNA plasmid (Evorgen) with Lipofectamine 2000 (Invitrogen) according to a manufacture's instruction [23] (link), [24] (link). G418 resistant transfectants were isolated as pHyPer stably transfected cells. Cellular fluorescence was assessed by a fluorescent microplate reader (SpectraMax Paradigm, Molecular Devices, YFP: Ex 497 nm, Em 525 nm, CFP: Ex 434 nm, Em 525 nm). YFP to CFP excited (497/434) ratio was calculated as intracellular H2O2 level [23] (link), [25] (link).
5
Extracellular ROS Scavenging by CeNP-PEG
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HK-2 cells were treated with100 μM of H2O2 for 2 h with the presence of the CeNP-PEG at different concentrations (0, 0.25, 0.50 and 1.00 μg/mL) or vehicle. The effect of CeNP-PEG on extracellular ROS scavenging was investigated by DCFH-DA. Briefly, cells were loaded with CM-H2-DCFDA reagent (Invitrogen) by incubating cells with 5 μM of probe solution in PBS for 20 min at 37 °C. Then, the fluorescence microscopy was visualized using a Carl Zeiss LSM710 confocal microscope, and processed using Photoshop software (Adobe Systems, Inc., San Jose, CA, USA).
6
Quantifying Cellular H2O2 in Skin
7
Intracellular ROS Production Assay
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The DCFDA assay for intracellular ROS production has been described previously [33 (link)]. Briefly, cells were loaded with CM-H2-DCFDA reagent (Invitrogen) by incubating cells with a 5 µM probe solution in PBS for 20 min at 37 °C. Basal fluorescence was then read to provide a well-background control and test conditions added to start the assay. Fluorescence intensity was monitored for 12 h.
8
Measuring Cellular Hydrogen Peroxide
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Cellular H2O2 was assayed using the CM-H2DCFDA reagent (Invitrogen) according to the manufacturer’s instructions.
9
Measuring Sperm H2O2 Permeability
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Hydrogen peroxide permeability of human sperm samples was measured by a fluorescence method using the CM-H2DCFDA reagent (Invitrogen, Carlsbad, CA, USA). Briefly, sperm cells were washed in PBS and centrifuged at 1000× g for 15 min. The cell pellet was resuspended in PBS and CM-H2DCFDA reagent was added at 5 mM final concentration and incubated for 1 h at room temperature. Thereafter, sperm cells were centrifuged again and the pellet resuspended in PBS. Cells in different experimental conditions (untreated (control) and treated with 100 μM HgCl2) were incubated with 50 μM H2O2; cellular H2O2 levels were detected over 5 min using a CLARIOstar® microplate reader (BMG LABTECH, Ortenberg, Germany). The initial rate constant of H2O2 uptake (k) was obtained by setting the time course light scattering with a single exponential equation (GraphPad Prism 4.00, 2003).
10
Quantifying Oxidative Stress in Cardiomyocytes
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ROS levels were detected using CM-H2DCFDA reagent
(Invitrogen) as per the manufacturer’s instructions. Briefly, primary
cultures of cardiomyocytes were treated with H2O2 (50
μM) in the presence or absence of HKL (10 μM) for 15 min. Cells
were stained with CM-H2DCFDA. Cells were acquired by FACSCalibur and
analyzed with use of FlowJo. The mean fluorescence intensity of cells positive
for CM-H2DCFDA staining was determined.
(Invitrogen) as per the manufacturer’s instructions. Briefly, primary
cultures of cardiomyocytes were treated with H2O2 (50
μM) in the presence or absence of HKL (10 μM) for 15 min. Cells
were stained with CM-H2DCFDA. Cells were acquired by FACSCalibur and
analyzed with use of FlowJo. The mean fluorescence intensity of cells positive
for CM-H2DCFDA staining was determined.
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