The cellular ROS content was measured using a fluorescence‐based assay using the cell‐permeable CM‐H2DCFDA dye (Invitrogen) according to the manufacturer's instructions. Briefly, cells were plated in black wall, clear bottom 96‐well plates at 15,000 cells/well in DMEM complete media, and cultured for 24 h. An oxidative stress inducer, tert‐butyl hydrogen peroxide (tBHP) (Sigma‐Aldrich) was used in some experiments to increase oxidative stress, as indicated. For the assay, cells were transferred to FluoroBrite media (Invitrogen) and exposed to 10 µM CM‐H2DCFDA for 30 min at 37℃. After washing with PBS, fluorescence was measured using a CLARIOstar plate reader at Ex/Em of 492–495/517–527 nm. The fluorescence intensity was normalized to the total protein from the same wells measured by the Bradford method. The results presented for ROS measurements are representative of three independent experiments.
Tert butyl hydrogen peroxide
Manufactured by Merck Group
Sourced in United States
Tert-butyl hydrogen peroxide is a colorless liquid chemical compound. It serves as an oxidizing agent and is commonly used in various industrial processes and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Tempol, by Merck Group (2 mentions)
Fluorobrite media, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti erp57, by Enzo Life Sciences (1 mentions)
Anti ledgf p75, by Fortis Life Sciences (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Catalase, by Merck Group (1 mentions)
Doxorubicin hydrochloride, by Merck Group (1 mentions)
Leibovitz s l 15 media, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Lipid peroxidation mda assay kit, by Abcam (1 mentions)
Pierce bicinchoninic acid protein kit, by Thermo Fisher Scientific (1 mentions)
Dcfda assay kit, by Abcam (1 mentions)
10 protocols using tert butyl hydrogen peroxide
1
Quantifying Cellular Oxidative Stress
2
Quantifying Cellular ROS Levels
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ROS was quantified using 10 µM chromomethyl 2′,7′ –dichlorofluorescin diacetate (CM-H2DCFDA), fluorescence (Sigma-Aldrich, D6883), 5 µm MitoSox red (Thermo Fisher Scientific, M36008), and 10 µM dihydroethidium fluorescence (DHE) (Thermo Fisher Scientific, D11347) and normalized to cell number. 50 µM tert-butyl hydrogen peroxide (TBHP) (Sigma Aldrich, 416665) was used as a positive control for DCFDA assay. ROS was quantified in the activated PBMCs from NGT subjects and lean subjects after fatty acid treatments (± Tre and ± Temp).
3
Quantifying Cellular ROS Production
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General cellular ROS production was assessed using DCF-DA (Abcam, Cambridge, United Kingdom). DCF-DA is a nonfluorescent compound that is membrane permeable and becomes highly fluorescent upon oxidation by ROS. BAECs were seeded in a clear-bottom 96-well plate at a concentration of 4–6 × 104 cells/well and allowed to attach overnight. On the day of the experiment, cells were washed once with PBS and stained with 25 μM DCF-DA in buffer and incubated for 45 minutes at 37°C. Cells were washed once with PBS and 100 μL of phenol-free media was added with treatment. Sildenafil 5 μM or placebo was added 5 minutes before irradiation. Cells were irradiated with 10 Gy and the fluorescent signal was read at excitation of 485 nm and at emission of 535 nm. tert-Butyl hydrogen peroxide (Sigma) was used as positive control according to the manufacturer’s instructions.
4
Quantifying Reactive Oxygen Species in Microglia
5
Assessing Prostate Cancer Cell Lines
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The metastatic prostate cancer cell lines DU145 (Cat.# HTB-81) and PC3 (Cat.# CRL-1435), the K-ras transformed prostate epithelial cell line RWPE-2 (Cat.# CRL-11610), derived from the normal prostate cell line RWPE-1, and the osteosarcoma cell line U2OS (Cat.# HTB-96) were purchased from the American Type Culture Collection (ATCC). Cells were cultured as recommended by the suppliers in a humidified incubator with 5% CO2 at 37°C. The following antibodies were used: mouse monoclonals anti-β-actin (1:5000, Sigma-Aldrich) and anti-ERp57 (1: 200, Enzo Life Sciences); rabbit polyclonal anti-LEDGF/p75 (1:1000, Bethyl laboratories Inc); goat polyclonal anti-Lamin B antibody C-20 (1:1000. Santa Cruz Biotechnology); human autoantibody to topoisomerase I (1:100, Topo I), a kind gift from Dr. Eng M Tan (Scripps Research Institute, La Jolla, CA); anti-LEDGF/p75 rabbit polyclonal antibody Scripps-Ab5087(1:1000), also donated by Dr. Eng M. Tan; and horseradish peroxidase (HRP)-labeled secondary IgG antibodies (1:5000, ThermoFisher Scientific). Tert-butyl hydrogen peroxide (TBHP), an organic peroxide, was purchased from Sigma-Aldrich. STS and N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-amc, fluorogenic caspase-3/7 substrate) were purchased from Axxora. The broad caspase inhibitor benzylocarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) was purchased from Biomol International.
6
Quantifying Cellular ROS Production
7
Quantifying Intracellular ROS Levels
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Intracellular ROS formation was assessed by measuring the fluorescence of DCF, the oxidation product of the non-fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH2-DA). Briefly, cells were grown in 96-well plates at a density of 2.0×105 cells per mL were treated with the IC25 and IC50 of AgNPs and G-AgNPs for 24h. Three hours prior to completion of treatment, 150 µM tert-butyl hydrogen peroxide (Sigma-Aldrich®, USA) was used as positive control. Then, cells were incubated with 10 µM DCFH2-DA (Merck Millipore®, USA) for 30 minutes at 37 °C, in the dark. Live cells were then imaged with filter set appropriate for fluorescein (FITC) using a fluorescence microscope OLYMPUS IX51 (Tokyo, Japan). The oxidation of DCFH2-DA was detected by the increase in fluorescence which is proportional to the amount of intracellular ROS generated. The intracellular mean fluorescence intensity was quantified using the ImageJ software (National Institutes of Health, USA).
8
Oxidative Stress Assays for Cells
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DCFDA assay kit (ab113851) and Lipid Peroxidation (MDA) assay Kit (ab118970) were purchased from abcam, USA. OxyIHC Oxidative Stress Detection Kit (S7450), Ascorbate, tert-butyl hydrogen peroxide, N-acetyl cysteine, catalase, SOD, and doxorubicin hydrochloride were procured from Sigma Aldrich, St. Louis, USA. All the other media samples including pierce bicinchoninic acid protein kit, DMEM/F-12, F-12 K, leibovitz’s L-15 media, FBS, and trypsin-EDTA (0.25%) were procured from Thermofisher, USA.
9
Quantification of Cellular Reactive Oxygen Species
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ROS was quantified using 2′,7′ –dichlorofluorescin diacetate (DCFDA) fluorescence (Sigma-Aldrich, D6883, St Louis, MO, USA) normalized to cell number [14 (link),17 (link)]. Twenty-five mM glucose (Sigma Aldrich, D8270) and 50 µM tert-butyl hydrogen peroxide (TBHP) (Sigma Aldrich, 416665) were used as positive controls. The free radical scavenger Tempol (50 µM; Sigma Aldrich SML0737) was used to manipulate ROS generation and served as a control [14 (link)].
10
Measuring Mitochondrial ROS with DCFDA
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ROS or mitochondrial ROS was assessed by 2’,7’ –dichlorofluorescin diacetate (DCFDA) fluorescence (Sigma- Aldrich, D6883), or MitoTEMPO (Santa Cruz Bitoechnology, sc-221945) normalized to cell number (Bharath et al., 2014 (link), Bharath et al., 2015 (link)). Treatment with 100 mM glucose (Sigma Aldrich, D8270) and 50 μM tert-butyl hydrogen peroxide (TBHP) (Sigma Aldrich, 416665) was the positive control. The free radical scavenger Tempol (10 μM; Sigma Aldrich SML0737) was used to manipulate ROS generation (Bharath et al., 2017a (link), Dikalova et al., 2010 (link)).
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