The intracellular ATP levels were measured using the ATPlite Luminescence ATP Detection Assay System (Perkin Elmer, Waltham, MA, USA). The ATP levels of the treated cells were normalized to the mean value obtained from untreated cells. Cytosolic and mitochondrial ROS were detected by flow cytometry after loading the cells with 5 μM DCFH-DA [2-(2,7-dichloro-3,6-diacetyloxy-9H-xanthen-9-yl)-benzoic acid] (Cayman Chemical, Ann Arbor, MI, USA) and 2 μM MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher), respectively. Mitochondrial membrane potential was measured by the fluorescent intensity of TMRE (Tetramethylrhodamine, ethyl ester) (Thermos Fisher) using flow cytometry after staining the cells with 50 nM TMRE for 15 min at 37 °C.
Dcfh da
Manufactured by Cayman Chemical
Sourced in United States
DCFH-DA is a fluorogenic probe used for the detection and quantification of reactive oxygen species (ROS) in biological samples. It is a cell-permeable compound that, upon oxidation by ROS, becomes the highly fluorescent dye DCF. The core function of DCFH-DA is to serve as a versatile tool for measuring intracellular ROS levels.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus 6, by Media Cybernetics (5 mentions)
Spectramax gemini em, by Molecular Devices (2 mentions)
Microplate reader, by PerkinElmer (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Atplite luminescence atp detection assay system, by PerkinElmer (1 mentions)
Hank s solution, by Merck Group (1 mentions)
Mts assay, by Promega (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Facscalibur, by BD (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Eclipse c1, by Nikon (1 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
24 protocols using dcfh da
1
Intracellular ATP and ROS Measurement
2
Intracellular ROS Measurement in RLECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular ROS level was measured using the fluorescent dye, dichlorofluorescein diacetate (H2-DCF-DA), and a nonpolar compound converted to a polar derivative (dichlorofluorescein) by cellular esterase after incorporation into cells [21 (link)]. RLECs (5 × 103) were cultured in 96-well plates for 120 hrs with DMEM and 5% FBS; 5.5 mM, 55 mM D-glucose, or 55 mM D-mannitol; and 50 μg/mL purified honey or 5 or 50 μg/mL propolis.
The medium was replaced with Hank's solution (Sigma) containing 10 μM DCFH-DA (Cayman Chemical, Ann Arbor, MI) and incubated for 10 min at room temperature. Intracellular fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using Spectra Max Gemini EM (Molecular Devices, Sunnyvale, CA).
The medium was replaced with Hank's solution (Sigma) containing 10 μM DCFH-DA (Cayman Chemical, Ann Arbor, MI) and incubated for 10 min at room temperature. Intracellular fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using Spectra Max Gemini EM (Molecular Devices, Sunnyvale, CA).
3
Measuring Intracellular Redox Levels and Cell Viability
4
Cellular Oxidative Stress Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cellular ROS levels were evaluated using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. In brief, WI-38 cells at a density of 5x105 cells/ml were treated with 10 µM DCFH-DA (Cayman Chemical Company) for 45 min in the dark at 37˚C. Afterwards, the images were captured under a fluorescence microscope (Olympus Corporation).
In brief, the cell medium was collected and the supernatant was collected after being centrifuged at 2,000 x g for 10 min at 4˚C. The levels of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and catalase (CAT) in the supernatants from WI-38 cells were assessed using their corresponding kits from Nanjing Jiancheng Bioengineering Institute (cat. nos. A003-1, A001-3 and A007-1, respectively) according to the manufacturer's protocols.
In brief, the cell medium was collected and the supernatant was collected after being centrifuged at 2,000 x g for 10 min at 4˚C. The levels of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and catalase (CAT) in the supernatants from WI-38 cells were assessed using their corresponding kits from Nanjing Jiancheng Bioengineering Institute (cat. nos. A003-1, A001-3 and A007-1, respectively) according to the manufacturer's protocols.
5
Intracellular ROS Measurement in Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of intracellular ROS were determined by a 2′,7′-dichlorodihydro-fluorescein diacetate assay (DCFH-DA, Cayman). Briefly, spheroids and their adherent cells were exposed to a HA/SF (3 × 106 cells/400 μL) mixture at 37 °C for 16 h. The washed cells were co-treated with 0.1% collagenase and 10 μM DCFH-DA, respectively, at 37 °C for 30 min. Then, the cells were evaluated by flow cytometry using a MACS Quant analyzer (Miltenyi Biotec, Gladbach, Germany).
6
Quantifying Cellular Free Radicals Using DCFH-DA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Measurement of the free radical level was carried out using fluorescent indicator 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Cayman Chemical Company), as described previously [45 (link)]. DCFH-DA is intracellularly deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH) and then oxidised to a fluorescent compound, 2′,7′-dichlorofluorescein (DCF). SH-SY5Y and BV2 cells were incubated in DCFH-DA (10 μM) solution in HBSS with 20 mM Hepes (pH 7.4) and 0.02% Pluronic for 50 min at 37 °C in the dark. Then, the cells were washed three times, and the DCF fluorescence was measured using a microplate reader FLUOstar Omega (Ortenberg, Germany) at 485-nm excitation and 538-nm emission wavelengths. After determining the baseline fluorescence of the cells incubated in HBSS, the change in fluorescence after the addition of the test compounds was recorded 3 h after treatment. The results of fluorescence measurements are presented as percent of corresponding control.
7
Measuring Cellular Reactive Oxygen Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular ROS levels were estimated using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) purchased from Cayman Chemical (85155, Ann Arbor, MI, USA). The cells were trypsinized and mixed with 1% FBS in PBS containing 10 µM DCFH-DA for 15 min, while protecting it from light. The fluorescent cells were analyzed on a FACSCaliburTM flow cytometer (Becton-Dickinson, San Diego, CA, USA).
8
Intracellular ROS Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of intracellular ROS were measured using DCFH-DA (Cayman Chemical, Ann Arbor, MI, USA). The NR8383 cells were incubated with 10 µmol/L DCFH-DA for 30 min at 37 °C. The Image-Pro Plus 6.0 software package (Media Cybernetics, Rockville, MD, USA) was used to quantify the integral optical density (IOD) of ROS fluorescence based on the images acquired by the fluorescence microscope.
9
Monitoring Intracellular ROS Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The production of ROS was monitored by fluorescence microscopy using DCFH-DA (Cayman Chemicals), following the manufacturer’s instructions. This dye is a stable compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield DCFH, which is trapped within cells. Hydrogen peroxide or low-molecular-weight hydroperoxides produced by cells oxidize DCFH to the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF). Thus, the intensity of the fluorescence is proportional to the amount of peroxide produced by the cells. For this assay, cells (104) in 96-well plates were loaded with 10 μM DCFH-DA for 30 min at 37°C in the dark, washed twice with PBS, detached with trypsin, and washed in PBS. After centrifugation, the cell pellet was suspended in 200 μl of PBS. The cells were observed under a fluorescence microscope (Leica). At the same time, fluorescence intensity of the cell was measured using a microplate reader (BioTek Synergy) with excitation at 485/20 nm and emission at 528/20 nm.
10
Intracellular ROS Measurement in hVICs
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!