Changes in the intracellular reactive oxygen species (ROS) levels were measured using the Intracellular ROS Assay Kit (OxiSelectTM, Cell Biolabs, San Diego, CA, USA) in accordance with the manufacturer’s instructions. The changes in dichlorofluorescein (DCF) fluorescence intensity were measured at wavelengths 485/520 nm using FLUOstar Optima.
Intracellular ros assay kit
Manufactured by Cell Biolabs
Sourced in United States
The Intracellular ROS Assay Kit is a fluorometric tool designed to measure the levels of reactive oxygen species (ROS) within cells. It provides a convenient and sensitive method for detecting and quantifying intracellular ROS production.
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Lab products found in correlation
Fluorometric plate reader, by Thermo Fisher Scientific (2 mentions)
Fv1200, by Olympus (1 mentions)
Ly294002, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Mcc950, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Eagle s minimum essential medium, by Welgene (1 mentions)
Hanks balanced salt solution (hbss), by Welgene (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Dulbecco s phosphate buffered saline pbs, by Welgene (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Welgene (1 mentions)
10 protocols using intracellular ros assay kit
1
Intracellular ROS Quantification
2
ROS Quantification in Bacterial Cultures
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Intracellular reactive oxygen species (ROS) were measured using an intracellular ROS assay kit (Cell Biolabs, Inc., San Diego, CA, USA). Cultured bacteria were incubated with 100 μM 2′, 7′-dichlorodihydrofluorescine diacetate (DCFH-DA) for 1 h at 37 °C in the dark, to preload them with the DCHF-DA probe. They were then washed with PBS and resuspended in a 60Φ cell culture dish and exposed to F-mode, IZ + F mode or IZ + F + C mode treatment, with stirring for 120 min. After exposure, the cells were harvested and transferred to black cell culture fluorometric 96-well plates and their DCF fluorescence intensity was measured at excitation wavelength 480 nm, emission wavelength 530 nm and 530 nm cutoff. For DCF fluorescence images, bacterial suspensions were loaded on glass slides after incubation with DCFH-DA for 1 h, and exposed to F-mode, IZ + F mode and IZ + F + C mode, respectively, for 120mins. DCF fluorescence signals were detected with a confocal microscope (Olympus FV1200, Japan).
3
Evaluating Bone and Immune Markers
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Rabbit polyclonal antibody (pAb) against ALP and mouse monoclonal antibody (mAb) against OPN and OCN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit pAbs against NLRP3, caspase 1, IL-1β, pAkt, Akt and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Intracellular ROS assay kit was purchased from Cell Biolabs (San Diego, CA). MCC950 (NLRP3 inflammasome inhibitor), N-acetylcysteine (NAC, ROS inhibitor), and LY294002 (PI3K/Akt inhibitor) and other chemicals were purchased from Sigma (Temecula, CA).
4
ROS Production Quantification Protocol
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ROS production was analyzed according to the manual of the Intracellular ROS
Assay Kit (Cell Biolabs, San Diego, CA). Cells were cultured in a 96-well cell
culture plate and treated with each concentration of OCE (1, 5, 10, 50, or 100
µg/mL) plus 10 µM 5-FU for 48 hours. Next, the cells pretreated with 100 µM
2′,7′-dichlorofluorescein diacetate (DCFH-DA) were incubated for 60 minutes at
37°C. The cell fluorescence was read on a fluorometric plate reader (Thermo
Fisher Scientific, Inc, Waltham, MA) at 480/530 nm. ROS production was
determined by comparison with the predetermined DCF standard curve.
Assay Kit (Cell Biolabs, San Diego, CA). Cells were cultured in a 96-well cell
culture plate and treated with each concentration of OCE (1, 5, 10, 50, or 100
µg/mL) plus 10 µM 5-FU for 48 hours. Next, the cells pretreated with 100 µM
2′,7′-dichlorofluorescein diacetate (DCFH-DA) were incubated for 60 minutes at
37°C. The cell fluorescence was read on a fluorometric plate reader (Thermo
Fisher Scientific, Inc, Waltham, MA) at 480/530 nm. ROS production was
determined by comparison with the predetermined DCF standard curve.
5
Intracellular ROS Measurement in RPEs
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The intracellular ROS accumulation in RPEs was measured by 2׳, 7׳-dichlorodihydro fluorescin diacetate (DCFH-DA) using an intracellular ROS assay kit (Cell Biolabs, San Diego, CA). ARPE-19 cells were pre-loaded with DCFH-DA by exposure to freshly prepared 100 µM DCFH-DA in the culture medium for 30 min in a 37 °C incubator. Then the DCFH-DA loaded cells were treated with 150 μg/mL oxLDL alone or combined with 30 μM E3330 for 48 h. DCFH fluorescence of the cell lysate was measured with excitation and emission settings of 485 and 530 nm by a microplate reader.
6
ROS Production in Cell Culture
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ROS production was performed according to the protocol of the intracellular ROS assay kit (Cell Biolabs, Inc., San Diego, CA, USA). Cells were cultured in a 96-well cell culture plate and treated with several concentrations of SM (1, 5, 10, 50 or 100 μg/ml) with 10 μM 5-FU for 48 h. Next, the cells pretreated with 1 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were incubated for 60 min at 37°C. After a brief incubation, the cell fluorescence was read on a fluorometric plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 480/530 nm. ROS production was determined by comparison with the predetermined 2′,7′-dichlorofluorescein (DCF) standard curve.
7
Quantifying Neutrophil Activation in Murine Colitis
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We quantified the extent of neutrophil activation by measuring MPO activity and ROS production in the colon of mice, respectively. Briefly, colons were dissected, rinsed with cold saline, and cut into small pieces. Samples were homogenized in 50 mM phosphate buffer and centrifuged at 12,000 ×g at 4°C for 20 min. We assayed MPO activity, an enzyme occurring nearly exclusively in neutrophils, using a commercial kit (BioVision, CA, USA), according to the manufacturer's recommended protocol. Additionally, we determined the levels of ROS in the colonic homogenates by using the Intracellular ROS assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer's instructions. Briefly, the lysates were incubated with 50 μM 2′,7′-dichlorodihydrofluoresceindiacetate (DCFH-DA) at 37°C for 1 h. Fluorescence intensities were measured by a fluorescence plate reader at 480 nm excitation/530 nm emission.
8
Intracellular ROS Measurement Protocol
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Intracellular ROS levels were measured by using intracellular ROS assay kit (Cell Biolabs, San Diego, CA). Briefly, ARPE-19 cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in the culture medium for 30 min at 37°C. The cells were assessed by using flow cytometry at 488 and 525 nm wavelengths. DCFH fluorescence of the cell lysate was captured and quantified by ImageJ software (NIH). The average fluorescence intensity was analyzed from five fields for each treatment.
9
Evaluating Antioxidant Efficacy of OC Extracts
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To confirm the antioxidant efficacy of three OC extracts on the generation of
intracellular ROS due to oxidative stress, intracellular ROS was measured using
the Intracellular ROS assay kit (Cell Biolabs, San Diego, CA, USA) according to
the proposed method. Briefly, ARPE-19 cells were plated on a 96-well plate
(1×104 cells/well) and incubated for 24 h in the
CO2 incubator. The cells were pre-treated with 2 mg/mL OC
extracts (hot water, ethanol, and methanol) for 1 h and stimulated with 300
μM H2O2 for 24 h. The cells were then treated with
DCF-DA (100 μM) and incubate for 1 h. The culture medium was completely
removed, washed twice with 1× PBS, and 100 μL of serum/phenol
red-free DMEM/F12 medium and 100 μL of cell lysis buffer (2×) were
added and incubated for 5 min. Finally, 150 μL of cell lysates were
transferred to a black plate and measured at 485 nm and 530 nm wavelengths using
a microplate reader.
intracellular ROS due to oxidative stress, intracellular ROS was measured using
the Intracellular ROS assay kit (Cell Biolabs, San Diego, CA, USA) according to
the proposed method. Briefly, ARPE-19 cells were plated on a 96-well plate
(1×104 cells/well) and incubated for 24 h in the
CO2 incubator. The cells were pre-treated with 2 mg/mL OC
extracts (hot water, ethanol, and methanol) for 1 h and stimulated with 300
μM H2O2 for 24 h. The cells were then treated with
DCF-DA (100 μM) and incubate for 1 h. The culture medium was completely
removed, washed twice with 1× PBS, and 100 μL of serum/phenol
red-free DMEM/F12 medium and 100 μL of cell lysis buffer (2×) were
added and incubated for 5 min. Finally, 150 μL of cell lysates were
transferred to a black plate and measured at 485 nm and 530 nm wavelengths using
a microplate reader.
10
Urolithin A Modulates Neuroblastoma Cell Viability
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The SK-N-MC human neuroblastoma cell line was purchased from American Type Culture Collection (Rockville, MD, USA). Eagle's minimum essential medium (EMEM), trypsin-EDTA, antibiotics, Dulbecco's phosphate-buffered saline (PBS), and Hank's balanced salt solution (HBSS) were purchased from WelGENE (Daegu, Republic of Korea). Fetal bovine serum (FBS), urolithin A (UA), and general reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell counting Kit-8 (CCK-8) assay reagents were purchased from Dojindo Molecular Technologies (Gaithersburg, MD, USA), and the intracellular ROS assay kit was purchased from Cell Biolabs (San Diego, CA, USA). Anti-Bax (#2772), anti-total p38 (#9212), anti-caspase-9 (#9502), anti-caspase-3 (#9665), anti-PARP (poly (ADP-ribose) polymerase, #9532), anti-mouse IgG horseradish peroxidase [HRP]-conjugated antibody (#7076), anti-rabbit IgG HRP-conjugated antibody (#7074), and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (#5633) were purchased from Cell Signaling Technology (Danvers, MA, USA).
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