Hydrogen peroxide-induced oxidative stress was assessed using dihydroethidium (DHE, Sigma-Aldrich) and CellROX green reagent (Life Technologies, Grand Island, NY). After exposure to 500 μM hydrogen peroxide for 6 h, 10 μM DHE or 5 μM CellROX green reagent was added to each cell on 4 chamber slides (Thermo Fisher Scientific) and incubated with DHE for 15minutes or with CellROX green reagent for 30 min at 37°C in DMEM. Cells were then fixed in 4% paraformaldehyde and mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Images were obtained using a confocal laser scanning microscopy (LSM 710; Carl Zeiss, Oberkochen, Germany). Fluorescence intensities were measured using ImageJ software (http://rsb.info.nih.gov/ij/ ). For superoxide quantification, cells were treated with DHE dye as above and 1.0 μg/mL Hoechst 33342 (Wako Pure Chemical Industries) in black walled clear bottom 96 well plates (Corning Life Sciences, Tewksbury, MA) for 15 min and were scanned in a fluorescent plate reader (PerkinElmer Ensight: Perkin-Elmer Inc., Wellesley, MA) at λex = 535 nm, λem = 610 nm for DHE, and λex = 350 nm, λem = 460 nm for Hoechst 33342. The DHE fluorescence intensity per cell was determined by dividing DHE fluorescence by the Hoechst 33342 fluorescence.
Cellrox green reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Italy, Germany, Australia, Spain
The CellROX Green Reagent is a fluorescent dye used to detect reactive oxygen species (ROS) in live cells. It is a cell-permeant dye that exhibits green fluorescence upon oxidation by ROS, providing a quantitative measure of oxidative stress within the cell.
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Lab products found in correlation
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (12 mentions)
Facscanto 2, by BD (8 mentions)
Facscalibur, by BD (8 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Mitosox red, by Thermo Fisher Scientific (7 mentions)
Lsm 700, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Menadione, by Merck Group (5 mentions)
Lsm 510, by Zeiss (5 mentions)
Opti mem, by Thermo Fisher Scientific (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Propidium iodide, by Merck Group (4 mentions)
Eclipse ti inverted microscope, by Nikon (3 mentions)
N acetyl cysteine (nac), by Merck Group (3 mentions)
N acetylcysteine nac, by Merck Group (3 mentions)
Trolox, by Merck Group (3 mentions)
Accuri c6, by BD (3 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (3 mentions)
382 protocols using cellrox green reagent
1
Quantitative Assessment of Oxidative Stress
2
Assessing Cellular Apoptosis and Oxidative Stress
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INS-1E cells were seeded onto glass cover slips and cultured in the presence of test substances as indicated in each experiment. After incubation cells were fixed with 4% formalin.
For TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, fixed cells were permeabilized with 0.1% Triton X-100 in PBS and fractionated DNA was detected using the In Situ Cell Death Detection Kit (Roche Diagnostics, Basel, Switzerland). Nuclei were stained with 0.1 μg/ml 4’6-diamidino-2-phenylindole (DAPI). The percentage of TUNEL-positive cells was evaluated by counting at least 300 cells/condition/experiment.
For TXNIP protein detection, the cells were permabilised with 0.2% Triton X-100, blocked with 10% FCS-PBS and incubated overnight with anti-TXNIP antibody (1:100; rabbit; Abcam) followed by incubation with anti-rabbit IgG-Alexa Fluor488 for 1h. The nuclei were stained with 1 μM TOPRO3.
ROS production was measured using CellROX® Green Reagent (Thermo Fisher Scientific). Menadione (100 μM, Sigma) was used as positive control. CellROX Green Reagent (5 μM, Invitrogen) was added during a second hour of incubation. An increase in CellROX® signal in the nuclei and cytosol indicates an increase in oxidative stress.
Fluorescence was detected using a confocal microscope (Leica).
For TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, fixed cells were permeabilized with 0.1% Triton X-100 in PBS and fractionated DNA was detected using the In Situ Cell Death Detection Kit (Roche Diagnostics, Basel, Switzerland). Nuclei were stained with 0.1 μg/ml 4’6-diamidino-2-phenylindole (DAPI). The percentage of TUNEL-positive cells was evaluated by counting at least 300 cells/condition/experiment.
For TXNIP protein detection, the cells were permabilised with 0.2% Triton X-100, blocked with 10% FCS-PBS and incubated overnight with anti-TXNIP antibody (1:100; rabbit; Abcam) followed by incubation with anti-rabbit IgG-Alexa Fluor488 for 1h. The nuclei were stained with 1 μM TOPRO3.
ROS production was measured using CellROX® Green Reagent (Thermo Fisher Scientific). Menadione (100 μM, Sigma) was used as positive control. CellROX Green Reagent (5 μM, Invitrogen) was added during a second hour of incubation. An increase in CellROX® signal in the nuclei and cytosol indicates an increase in oxidative stress.
Fluorescence was detected using a confocal microscope (Leica).
3
Quantifying Cellular Oxidative Stress
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For neurons, 5 μM CellROX Green Reagent (Invitrogen, C10444) and 10 μg/ml Hoechst-33342 (nuclear counter staining) were added to cell medium. Cells were then incubated for 30 min at 37°C in a humidified 5% CO2 incubator, followed by washing 3 times with pre-warmed PBS. The fluorescence intensity was measured using Cytation 5 (BioTek) at 520 nM for CellROX and 461 nm for Hoechst-33342. The individual ROS level was calculated by normalizing CellROX intensity (ROS level) to corresponding Hoechst-33342 intensity (overall cell density).
Astrocytes were incubated with 5 μM CellROX Green Reagent (Invitrogen, C10444) for 30 min at 37°C in a humidified 5% CO2 incubator. After three washes with pre-warmed PBS, astrocytes were trypsinized then pelleted by centrifuging and resuspended in 150 μl of PBS with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signal was detected by MACSQuant Analyzer Flow Cytometry (Mitenyi Biotec) and analyzed with FlowLogic software.
Astrocytes were incubated with 5 μM CellROX Green Reagent (Invitrogen, C10444) for 30 min at 37°C in a humidified 5% CO2 incubator. After three washes with pre-warmed PBS, astrocytes were trypsinized then pelleted by centrifuging and resuspended in 150 μl of PBS with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signal was detected by MACSQuant Analyzer Flow Cytometry (Mitenyi Biotec) and analyzed with FlowLogic software.
4
Quantifying Cellular Oxidative Stress
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CellROX® Green reagent (Thermo Fisher Scientific) was used to measure oxidative stress. Cells were incubated with 5 µM CellROX Green reagent for 30 min, fixed with 3.7% formaldehyde, nuclei, and F-actin were labeled with DAPI and Alexa Fluor 568 Phalloidin (Thermo Fisher Scientific), respectively. Images were acquired with a fluorescence microscope equipped with an ApoTome.2 system. CellROX signal was quantified with a Safire 2 microplate reader (Tecan). Signals of the microplate reader were normalized to soluble protein concentration after lysis with RIPA buffer.
5
Intracellular ROS Levels in Macrophages
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Levels of intracellular ROS were determined using CellROX® Green Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Elicited macrophages from WT and Nrf2−/− mice were exposed to LGM2605 (50 µM and 100 µM) 4 h prior to asbestos challenge (20 µg/cm2) and cells were harvested at 24 h post asbestos exposure. Asbestos-treated and untreated cells were incubated with 5 μM CellROX® Green Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37 °C after which cells were washed with phenol red free RPMI and imaged on a Nikon TMD fluorescence microscope (Nikon Diaphot TMD, Melville, NY, USA) equipped with a Hamamatsu ORCA-100 camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). All fluorescent cell images were acquired at the same exposure and offset settings using the MetaMorph acquisition software (Version 7.7, Molecular Devices, Downington, PA, USA). The fluorescent images of cells were processed and quantitated for CellROX® Green Reagent fluorescence by the use of ImageJ software (Fiji Version, National Institutes of Health, Bethesda, MD, USA). The intensity of cells in each field was integrated to obtain the total fluorescence intensity of a particular field. Scale bar = 20 μm.
6
Renal Tubular ROS Quantification
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The detectable amount of renal tubular ROS can be a surrogate for the severity of oxidative stress during CI-AKI. We tested ROS levels on frozen renal sections using CellROX Green Reagent from Thermo Fisher Scientific (Rockland, IL, USA); the signal emitted from CellROX Green Reagent will localize to the nucleus and mitochondria upon oxidation. This reagent has been used to estimate ROS [8 (link), 17 (link), 18 (link)] produced by molecules such as angiotensin II, lipopolysaccharide, menadione, and nefazodone. We permeabilized frozen sections in 0.5% Triton® X-100 for 10 min and incubated slides with CellRox (5 μM) for 30 min at 37°C. These slides were then washed in phosphate-buffered saline (PBS). DAPI (2 mg/mL) was used as the nuclear counterstain. After a PBS wash, we acquired images using fluorescence microscopes (Olympus BX51, Japan) and analyzed the results with ImageJ (V1.44, http://rsbweb.nih.gov/ij/ ) software, followed by image merging and synthesis of composite figures. The ROS-positive cells were counted at 400x magnification over 10 fields, with results described using percentages of total cells.
7
Measuring Oxidative Stress Response
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ROS levels were analysed using the fluorogenic probe CellROX® Green Reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). At the specified time points after radiation-based treatments, the CellRox reagent was added in the culture medium to a final concentration of 5 µM. After 30 min incubation in the dark at 37 °C, cells were washed twice with PBS and harvested. Samples were acquired on a flow cytometer (Navios EX, Beckman Coulter, Pasadena, CA, USA) and results were analysed using Kaluza Flow Cytometry Analysis v2.1 software (Beckman Coulter, Pasadena, CA, USA). Relative fluorescence intensities were obtained from the median fluorescence intensity of each histogram of the different experimental conditions.
8
Quantifying ROS-induced DNA Damage
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Cells were incubated in growth medium containing 2.5 μM CellRox Green reagent (Invitrogen, USA) for 30 min at 37 °C. ROS levels were quantitated by measuring Mean Fluorescence Intensity (MFI) in individual cells using flow-cytometric analysis on a BD-Accuri flow cytometer using the C6 software. TBHP (tert-butyl hydroperoxide, 100 μM) (458149, Sigma, USA) was used as positive control. 2.5 mM NAC (N-acetylcysteine) (A9165, Sigma, USA) was used as a ROS scavenger for 30 min prior to TBHP treatment. ROS dependent DNA damage was measured by treating cells with 50 µM H2O2 for 30 min in the presence or absence of caspase inhibitor z-VAD-fmk (Promega, USA) or ROS scavenger NAC. The DNA damage was confirmed using western blotting with the anti-γH2AX antibody.
9
Measuring Cellular Oxidation and Proliferation
10
Oxidative Stress Evaluation in Cells
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Cells were plated on glass slides in 6-well plates. The cells were treated with 20 μM IS and/or 25 ngml−1 TRAIL in the presence/absence 2 mM NAC for 24 h at 37°C. The cells were then stained with 5 μM CellROX™ Green Reagent (Invitrogen, Carlsbad, CA, USA) and incubating at 37°C for 30 min. The cells were washed with PBS and imaged on a Leica DMI 3000 B inverted microscope using a 40x objective or analyzed by Flow cytometry.
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