Intracellular ROS were detected in live cells using the Fluorometric Intracellular ROS kit (Sigma-Aldrich). Cells were harvested and resuspended in Dulbecco's phosphate buffered saline (Sigma) at 5×105 cells/ml. To 500 µl of cells in microcentrifuge tubes, 600 µl of Master Reaction Mix was added from the Fluorometric Intracellular ROS kit (Sigma-Aldrich) after which the cells were incubated in darkness under 5% CO2 at 37°C for 1 h. The fluorescence (λex=520 nm/λem=605 nm) was measured in a Modulus Fluorometer (Turner Biosystems) using the Green Module. An arbitrarily selected control cell line (C105) was used in every experiment as an internal, normalization control for between-experiment variation. The fluorescence is proportional to the amount of ROS present. In separate control experiments the fluorescence was shown to be quenched dramatically by addition of 100 mM mannitol, a colourless ROS trap that works by reacting with hydroxyl radicals (data not shown).
Fluorometric intracellular ros kit
Manufactured by Merck Group
Sourced in United States, Germany, Belgium
The Fluorometric Intracellular ROS Kit is a laboratory equipment product designed to measure the levels of reactive oxygen species (ROS) within cells. The kit utilizes a fluorescent probe that reacts with ROS, allowing for quantification of intracellular ROS levels.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s phosphate buffered saline, by Merck Group (3 mentions)
Facscalibur, by BD (3 mentions)
Dmi6000b, by Leica (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Annexin 5 apc, by Thermo Fisher Scientific (2 mentions)
2 2 azino bis, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Euroclone (2 mentions)
Envision fluorometric plate reader, by PerkinElmer (2 mentions)
Modulus fluorometer, by Promega (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Ab228549, by Abcam (1 mentions)
Hoechst, by Merck Group (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
Annexin 5 fitc pi detection kit, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
77 protocols using fluorometric intracellular ros kit
1
Quantifying Intracellular ROS Levels
2
Quantifying Intracellular ROS in BMDMs
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BMDMs were seeded in 24-well plates at a density of 2 × 105 cells per well, treated with opsonized RBCs (1:10) and intracellular ROS was determined with a fluorometric intracellular ROS kit (Sigma-Aldrich, MAK142). Stained cells were visualized and captured using Leica DMI6000B with Cy5 filter set. The ImageJ software was applied to analyze regions of interest (ROIs) over multiple cells (n > 50). The fluorescence intensity from all the ROIs was averaged, and the corresponding background average was subtracted to yield the signal intensity for each condition.
3
Quantifying ROS Levels in CMECs
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Total ROS levels in CMECs were measured using the Fluorometric Intracellular ROS Kit (MAK142; Sigma, USA) and mitochondrial ROS levels in CMECs were measured using a Mitosox Red Mitochondrial Superoxide Indicator (M36008; Invitrogen, USA) according to the manufacturer's instructions. The fluorescent signal intensity was measured with a Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Japan).
4
Measuring Cellular and Mitochondrial ROS
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Isolated neutrophils or LDGs were stained with ROS detection reagent, and the mean fluorescence intensity (MFI) was measured by flow cytometry. Cytosolic ROS production was measured by Fluorometric Intracellular Ros Kit (Sigma-Aldrich, USA), and mitochondrial ROS production was measured by MitoSOX (Invitrogen, USA) respectively.
5
Quantifying Intracellular ROS in EPCs
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Intracellular ROS levels were measured by a Fluorometric Intracellular ROS Kit (MAK142; Sigma‐Aldrich, USA). EPCs were seeded on fibronectin‐coated 12 mm cover glasses in a 24‐well plate. The EPCs were treated with the indicated concentration of FGF21 for 12 h and 600 µM H2O2 for 1 h. The cells were washed with PBS and incubated with ROS Detection Reagent at 5% CO2 and 37°C for 1 h. After 1 h, the samples were carefully washed with PBS and then stained with DAPI (1:1000; ab228549; Abcam, USA) for 15 min at room temperature. The cover glasses were mounted with mounting medium (Dako, USA), and images were captured with a laser confocal microscope (ZEISS LSM 880, ZEISS, Germany).
6
Quantifying Oxidative Stress in rBMSCs
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rBMSCs were seeded in 12-well plates in triplicate at a density of 2 × 104 cells/well. After routine culturing (with or without N-acetylcysteine, NAC: 5 mM) or miR-22 transfection, the cells were exposed to 0 or 6 Gy of IR, and analysis was performed at 24 h after X-ray exposure. For ROS staining, the cells were incubated with the Fluorometric Intracellular ROS Kit (Sigma-Aldrich, Merck, Germany) for 45 min (5% CO2, 37 ℃), gently washed with PBS 3 times, counterstained with 10 μg/ml Hoechst (Sigma-Aldrich, Merck) for 10 min, and then imaged under a fluorescence microscope. To determine the fluorescence intensity, the incubated cells were measured on a flow cytometer with the following parameters: λ ex = 640 nm and λ em = 675 nm. The results are shown as images and fluorescence intensity.
7
Quantifying Intracellular ROS in BMDMs
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BMDMs were seeded in 24-well plates at a density of 2 × 105 cells per well, treated with opsonized RBCs (1:10) and intracellular ROS was determined with a fluorometric intracellular ROS kit (Sigma-Aldrich, MAK142). Stained cells were visualized and captured using Leica DMI6000B with Cy5 filter set. The ImageJ software was applied to analyze regions of interest (ROIs) over multiple cells (n > 50). The fluorescence intensity from all the ROIs was averaged, and the corresponding background average was subtracted to yield the signal intensity for each condition.
8
Oxidative Stress and INS-1 Cell Viability
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The oxidative injury INS-1 cell model was established with H2O2 as described previously.30 (link) Briefly, INS-1 cells were inoculated with 1.5×104 cells/well in 96-well plates and were cultured for 72 h at 37°C with 5% CO2. H2O2 was added to the wells at a final concentration of 400 μM and the cells were incubated for 1 h. Then the cells were treated for 24 h in fresh media containing 10 nM of SCD (using the final concentration of DBAYL as the quantitative index). The cell proliferation rates were determined with MTT. Intracellular ROS levels were determined with fluorometric intracellular ROS kit (Sigma-Aldrich) using the method described previously.31 (link) For SCD (10 nM) treatment, the same doses of SeNPs, SC (210 nM, using the final concentration of SeNPs as the quantitative index), BAY55-9837, Ex-4, DBAYL (10 nM) or the same volume of PBS were used for controls.
For the receptor VPAC2 blocking experiments, cells were preincubated for 30 min at 37°C with 50 nM of a VPAC2-selective inhibitor PG99-465 prior to 10 nM SCD treatment.
For the receptor VPAC2 blocking experiments, cells were preincubated for 30 min at 37°C with 50 nM of a VPAC2-selective inhibitor PG99-465 prior to 10 nM SCD treatment.
9
Fluorometric Assay for Reactive Oxygen Species
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The generation of reactive oxygen species (ROS) was determined using the Fluorometric Intracellular ROS Kit (#MAK143, Sigma® Life Science, Darmstadt, Germany), following the manufacturer’s protocol. Cells were prepared in the same way and in the same density as for the previous assays. The results were measured in a 96-well plate (half area, transparent bottom, black walls) after 6 h of exposure to WWs from R. damascena and R. centifolia. The fluorometric reaction product proportional to the amount of ROS present was measured at λex = 490/λem = 520 nm (Bio-Tek Instruments Inc., Winooski, VT, USA).
10
Osteogenic Differentiation and ROS Regulation
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Annexin V-FITC/PI detection kit was purchased from BD Biosciences (San Jose, CA, USA). Fluorometric Intracellular ROS Kit and N-acetyl-L-cysteine (NAC) were from Sigma-Aldrich (St. Louis, USA). Information of primary antibodies is listed as follows: anti-Runx2 (1 : 500, Abcam), anti-Osterix (1 : 500, Abcam), anti-OPN (1 : 1000, Abcam), anti-BSP (1 : 1000, CST), anti-OCN (1 : 500, Abcam), anti-NADPH oxidase 4 (NOX4) (1 : 500, Abcam), anti-SOD2 (1 : 500, Abcam), anti-Caspase-3 (1 : 1000, CST), and anti-GAPDH (1 : 5000, Bioworld Technology Inc., USA). ALP staining and alizarin red were both from Cyagen (Guangzhou, China). For semiquantitative analysis, p-nitrophenyl phosphate (p-NPP) and 10% cetylpyridinium chloride were from Sigma-Aldrich (St. Louis, USA). Alexa Fluor 488 conjugated was from Jackson ImmunoResearch (USA). DAPI was from Sigma-Aldrich (St. Louis, USA). For scaffold fabrication, gelatin, carboxymethyl chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were all purchased from Aladdin (Shanghai, China). Calcein AM (4 mM) and Lipofectamine 3000 were from Thermo Fisher Scientific (USA).
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