Oxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein (DCF) diacetate. After incubation, a 150 µl suspension of erythrocytes was washed in Ringer solution and stained with DCF diacetate (Sigma, Schnelldorf, Germany) in Ringer solution containing DCF diacetate at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and washed two times in Ringer solution. The DCF-loaded erythrocytes were resuspended in 200 µl Ringer solution and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).
Dcf diacetate
Manufactured by Merck Group
Sourced in Germany
DCF diacetate is a laboratory reagent used for various analytical and research applications. It serves as a precursor compound in biochemical processes. The core function of DCF diacetate is to provide a controlled release of the active component, which is essential for specific experimental procedures. No further interpretation or extrapolation on the intended use is provided.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Fluoroskan ascent spectrofluorimeter, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tissue tearor, by Biospec (1 mentions)
Varioskan spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Multi detection microplate reader, by Agilent Technologies (1 mentions)
Ix81 fluorescence microscope, by Olympus (1 mentions)
7 protocols using dcf diacetate
1
Erythrocyte Oxidative Stress Evaluation
2
Oxidative Stress Quantification in Erythrocytes
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Oxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein (DCF) diacetate. After incubation, a 100 µl suspension of erythrocytes was washed in Ringer solution and stained with DCF diacetate (Sigma, Schnelldorf, Germany) in Ringer solution containing DCF diacetate at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and washed two times in Ringer solution. The DCF-loaded erythrocytes were resuspended in 200 µl Ringer solution and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).
3
Measuring Oxidative Stress by DCF Fluorescence
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The accumulation of ROS was determined by analysing dichlorofluorescein (DCF) fluorescence. For determination of ROS accumulation, DCF-diacetate, 0.5 µM (Sigma-Aldrich, Saint Louis, Missouri, USA) was applied to the protein extract of cortex and hippocampus. The free DCF-diacetate was readily converted into DCF, which is able to interact with peroxides (primarily H2O2) to form the highly fluorescent DCF. DCF fluorescence was quantified (excitation at 485 nm, emission at 530 nm) using a Fluoroskan Ascent spectrofluorimeter (Thermo Scientific, Waltham, USA), normalised for protein concentration.
4
Hepatic Oxidative Stress Evaluation
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Proteins were extracted from whole liver tissues using RIPA buffer and quantified using the Bradford assay (Nanjing Jiangcheng Bioengineering Institute). The SOD, MDA and GSH-Px content of liver tissues was determined using the kits obtained from Nanjing Jiangcheng Bioengineering Institute, according to the manufacturer's protocols. To estimate hepatic ROS level, liver tissues were harvested and immediately homogenized in PBS using a Teflon homogenizer (Tissue-Tearor; BioSpec Products Inc.). Briefly, 50 µl liver homogenate was mixed with 4.85 ml of 100 mmol/l potassium phosphate buffer (cat. no. 700621-5; Cayman Chemical) and incubated with 2′,7′-dichlorofluorescin (DCF) diacetate (Sigma-Aldrich; Merck KGaA) in methanol at a final concentration of 5 µmol/l for 15 min at 37°C. The hepatic ROS level was determined as the amount of 2′,7′-dichlorofluorescein (DCF) quantified from a DCF standard curve. The BCA protein assay kit (Thermo Fisher Scientific, Inc.) was used to measure protein concentration. The fluorescence of DCF was measured with a Varioskan spectrophotometer (Thermo Fisher Scientific, Inc.) at excitation/emission wavelengths 480/530 nm.
5
Antioxidant Assay of Thai Fruit Extracts
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The intracellular antioxidant activities of the crude extract of Thai fruits were quantified using a fluorescent probe dichlorodihydrofluorescein (DCF) diacetate (Sigma-Aldrich) to estimate the intracellular ROS production in the cell as previously described [20 (link)]. HEK-293 cells were seeded in a 12-well plate (5 × 104 cells/well) or 35-mm glass bottomed dishes (1 × 105 cells/dish) and treated with Thai fruit extracts (10 and 100 μg/ml) for 12 h. After 12 h, the cells were then incubated with 100 μM of H2O2 for 2 h and washed once with phosphate buffered saline (PBS), pH 7.4. Thereafter, 10 μM DCFH-DA was added and incubated with the cells at 37°C in the dark for 30 min. The fluorescence intensity of DCF was determined using Multi-Detection Microplate Reader (BioTek Instruments) with an excitation wavelength of 485 nm and emission wavelength of 530 nm. To monitor the ROS production in living cells, HEK-293 cells were visualized using single-line excitation (488 nm) of an IX-81 fluorescence microscope (Olympus) with a 40x (NA 1.4) objective lens (Olympus).
6
Oxidative Stress Assessment in Erythrocytes
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Oxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein (DCF) diacetate. After incubation, a 100 µl suspension of erythrocytes was washed in Ringer solution and stained with DCF diacetate (Sigma, Schnelldorf, Germany) in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and washed two times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µl Ringer solution and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).
7
Quantifying Hippocampal ROS Accumulation
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ROS accumulation was determined by analyzing 2',7'-dichlorofluorescein (DCF) fluorescence. DCF diacetate (0.5 µM) (Sigma-Aldrich) was applied to the hippocampus extract. DCF diacetate was readily converted into DCF, able to interact with peroxides (primarily H 2 O 2 ) to form fluorescent DCF. DCF fluorescence was quantified (excitation 485 nm, emission 530 nm) using a Fluoroskan Ascent spectrofluorimeter (Thermo Scientific, Waltham, USA), normalized for protein concentration.
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