DOX was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China) and dissolved in PBS. SYKT (1.02 g/ml) containing sanguis draconis, radix et rhizoma notoginseng, radix et rhizoma glycyrrhizae, radix angelicae sinensis, ginger, Rhizoma Dioscoreae, Poria cocos and Fructus Amomi., was purchased from Great Tao Pharmaceutical Co., Ltd (Yunnan, China). A mixture of Cremophor RH40 (BASF SE, Ludwigshafen, Germany) and dehydrated alcohol (1:1, w/w) was used to dissolve vitamin E (d, l-a-tocopherol; BASF SE) to a stock concentration. Further dilutions were made with water prior to injection at a concentration corresponding to 100 IU/kg for each animal. All drugs were prepared immediately prior to use, with sterile solvents and under sterile conditions. Mouse lymphoma (EL4) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Anti-CD34 (catalogue no. 551387) and CD44 antibodies (catalogue no. 561859) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit was purchased from Merck KGaA (Darmstadt, Germany). ROS detection reagents were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
Ros detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
ROS Detection Reagents are a suite of fluorescent probes designed to detect and quantify reactive oxygen species (ROS) in biological systems. These reagents provide a reliable and sensitive method for measuring various ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals, which are important indicators of cellular oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Cremophor rh40, by BASF (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Novocyte advanteon flow cytometer, by Agilent Technologies (1 mentions)
Facscanto 2, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dnase free rnase a, by Merck Group (1 mentions)
Annexin 5 pi apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Mda assay kit, by Abcam (1 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Mitotracker green mt green, by Thermo Fisher Scientific (1 mentions)
4 5 diaminofluorescein diacetate daf 2 da, by Thermo Fisher Scientific (1 mentions)
8 hydroxy 2 deoxyguanosine 8 ohdg, by R&D Systems (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using ros detection reagent
1
Therapeutic Evaluation of DOX and SYKT
2
Intracellular ROS Detection Assay
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Assessment of intracellular ROS was assessed using the ROS detection reagents (Thermo Fisher Scientific). In brief, 1 × 104 cells were harvested and cultured under the same conditions described earlier. The cells were then incubated with DCF-DA (2,7-dichlorofluorescin diacetate) (10 μM) for 30 mins at room temperature to detect ROS.
3
Mitochondrial ROS and Apoptosis Assay
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Mitochondria ROS levels were measured using the MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen) according to the manufacturer's protocol. Cellular ROS levels were measured using ROS Detection Reagents (Thermo Fisher) according to the manufacturer's protocol. Apoptosis assay was performed with an Annexin V‐FITC Apoptosis Detection Kit (Dojindo) according to the manufacturer's instructions. Stained cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent).
4
Assessing ROS Levels in TiO2-Exposed ADSCs
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ROS Detection Reagents (Cat#: C6827, Invitrogen) was used to detect ROS level of ADSCs cells. For this experiment a working solution of 5 µg/mL of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was prepared. Cultures were seeded with starting density of 8 × 104 per well in six-well plate and exposed to TiO2 for 3 days. Cells were then harvested and washed three times with PBS to remove TiO2 NPs from pellets, counted and 5 × 104 cells per well were placed to 96-well dish (each condition had triplicates). Then 100 µL of working solution was added to each well and incubated for 20 min. 100 µL of 20 mM NaN3 were then added to each well and incubated for 2 h. Fluorescence was read at 490 nm excitation and 520 nm emission.
5
Comprehensive Cell Apoptosis and Cycle Analysis
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For cell apoptosis assay, the Annexin V/PI Apoptosis Detection kit (AD-10, Dojindo Molecular Technologies, Japan) was used according to the manufacturer’s protocol.
For intracellular ROS detection, Reactive Oxygen Species (ROS) Detection Reagents (Invitrogen, #D399) was used according to the manufacturer’s protocol.
For cell cycle analysis, harvested cells were fixed with 75% ethanol overnight followed by staining with a phosphate-buffered saline (PBS) solution containing propidium iodide (50 μg/ml, Sigma) and 100 μg/ml DNase-free RNase A (Sigma). After 30 minutes of incubation, the samples were washed and resuspended in PBS with 0.5% FBS. FACS analyses were performed on a BD FACSCanto™ II (BD Biosciences, USA).
For intracellular ROS detection, Reactive Oxygen Species (ROS) Detection Reagents (Invitrogen, #D399) was used according to the manufacturer’s protocol.
For cell cycle analysis, harvested cells were fixed with 75% ethanol overnight followed by staining with a phosphate-buffered saline (PBS) solution containing propidium iodide (50 μg/ml, Sigma) and 100 μg/ml DNase-free RNase A (Sigma). After 30 minutes of incubation, the samples were washed and resuspended in PBS with 0.5% FBS. FACS analyses were performed on a BD FACSCanto™ II (BD Biosciences, USA).
6
Intracellular ROS Measurement in Hepatocytes
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The production of intracellular ROS was measured using the ROS detection reagents (Invitrogen) according to the manufacture’s instructions. In brief, primary hepatocytes were plated into 96-well plate and cultured in 21% O2 or 5% O2 conditions. At day 1, day 3, and day 5, the cells were washed twice with PBS and supplied with phenol red-free DMEM containing 10 μM DCFH-DA dye and then incubate for 30 min at 37 °C in dark. Cells were then washed with PBS for three times and the DCF fluorescence intensity was measured using microplate fluorescence reader (excitation wavelength: 488 nm and emission wavelength: 530 nm).
7
Measurement of Intracellular ROS in INS-1E Cells
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The production of intracellular ROS was measured using the ROS detection reagents (Invitrogen) according to the manufacturer’s instructions. In brief, INS-1E cells were plated into 96-well plates. After treatment with 0.4 mM PA for 24 h, the cells were washed twice with PBS and supplied with phenol red-free RPMI-1640 containing 10 μM DCFH-DA dye and then incubated for 30 min at 37 °C in the dark. The cells were then washed with PBS three times and the DCF fluorescence intensity was measured using microplate fluorescence reader (excitation wavelength: 488 nm and emission wavelength: 530 nm).
8
Quantifying Embryonic ROS Levels
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Reactive oxygen species (ROS) levels within embryos were measured using ROS Detection Reagents (Invitrogen), according to the manufacturer’s instructions. In this procedure, dihydrocalcein-acetoxymethylester freely permeated cell
membranes and was oxidized by ROS to emit green fluorescence. Embryo fluorescence was observed under a fluorescent digital microscope, and intensities of pixel fluorescence were quantified using ImageJ, as described above.
membranes and was oxidized by ROS to emit green fluorescence. Embryo fluorescence was observed under a fluorescent digital microscope, and intensities of pixel fluorescence were quantified using ImageJ, as described above.
9
Oxidative Stress Biomarkers Quantification
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Cells were crushed by an ultrasonic shaker prior to the detection of MDA, ROS and Ca2+. MDA was measured using the Lipid Peroxidation (MDA) Assay Kit (Abcam, Cat# ab118970). MDA is converted into MDA-TBA adduct by reaction with TBA in the sample. The conversion can then be measured calorimetrically at 532 nm. The intracellular ROS content was determined using ROS Detection Reagents (Invitrogen, Cat# D399). Following cells reaching at 100% confluence in black 96-well dark plates, ROS detection was carried out using the indicated treatments. Cells were then incubated with 25 µM H2DCFDA probes indicators in warm 1×PBS at 32 °C incubator for 30 min. Subsequently, ROS content was measured using a spectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
10
Neutrophil ROS Production Assay
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Isolated PMNs (1x105) were re-suspended in 50 μL of Hanks Balanced Salt Solution (HBSS+1% FBS) per well of a 96-uncoated serum well plate. Cell suspension was supplemented with Reactive Oxygen Species (ROS) Detection Reagents (Invitrogen) and stimulated with phorbol myristate acetate (40 nM), Br-LPS (0.03–3 pmol/mL), Ec-LPS (0.09–7.5 pmol/mL) in 50 μl HBSS+1% FBS or left untreated. The kinetics of ROS production was monitored with a Victor Perkin Elmer luminometer at 37C for 90 min.
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