The average level of ROS was measured using a ROS assay kit DCFH-DA (Beyotime Biotech, Nanjing, China). Approximately 3×105 cells/well were seeded in 6-well plates overnight followed by the treatment of SPIO-Serum for 24 h. DCFH-DA was diluted to a final concentration of 10 μM. The cells were collected and suspended in diluted DCFH-DA in the dark at 37°C for 30 min and washed 3 times with PBS, and then samples were analyzed using an Accuri C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Dcfh da
Manufactured by BD
Sourced in United States
DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) is a fluorogenic compound used as an indicator for the detection of reactive oxygen species (ROS) in biological systems. It is a cell-permeable dye that can be oxidized by various ROS, including hydrogen peroxide, superoxide, and hydroxyl radicals, to produce the fluorescent compound 2',7'-dichlorofluorescein. The intensity of the fluorescent signal is proportional to the level of ROS present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Dcfh da, by Merck Group (31 mentions)
Dcfh da, by Beyotime (24 mentions)
Dcfh da, by Thermo Fisher Scientific (8 mentions)
Facscalibur, by BD (8 mentions)
Facscalibur flow cytometer, by BD (7 mentions)
Flow cytometry, by BD (4 mentions)
Software, by FlowJo (3 mentions)
Accuri c6, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Facsaria, by BD (2 mentions)
Facsaria 3, by BD (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Ow cytometer, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Dcfh da, by MedChemExpress (2 mentions)
Summit software, by Agilent Technologies (1 mentions)
Cellquest pro software version 1, by BD (1 mentions)
C6 biosciences, by BD (1 mentions)
Cellquest software version 5, by BD (1 mentions)
77 protocols using dcfh da
1
Quantifying Intracellular ROS Levels
2
Measuring Intracellular ROS in H358 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of ROS in H358 cells was measured using a ROS sensitive fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, ThermoFisher). DCFH-DA can be oxidized to 2′,7′-dichlorofluorescein (DCF) by ROS and exhibits green fluorescence intensity.80 H358 cells were seeded on 25 × 25 mm microscope cover glass slips in BD Falon 60 × 60 mm tissue culture dishes for 72 h. Untreated cells were maintained as the negative controls whereas 10, 20 and 30% H2O2 solution in PBS was administered to cells for 15 minutes as positive controls.54 (link) H358 cells were also dosed with IC50 values of various complex as follows: 44+ (15 μM), 33+ (13 μM) and 22+ (1.7 μM) for 3 time periods of 2, 8, and 22 h. The cells were passaged and washed 3× in ice cold PBS then suspended in 10 mM DCFH-DA in PBS and incubated in the dark for 30 min. The levels of intracellular ROS were examined by confocal microscopy using long pass light filters and a 1.3 airy unit pinhole at 488/529 nm with a Zeiss axio-plane inverted fluorescence microscope.
3
Quantifying ROS and Pyroptosis in NPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ROS in the NPCs were quantified with dichloro-dihydro-fluorescein diacetate (DCFH-DA, 10 μM, S0033, Beyotime, Shanghai, China). After treatment with DCFH-DA (2 μl of DCFH-DA dissolved in 2 ml RPMI 1640) at 37°C for 20 min, the fluorescence was analyzed using a FACSVerse (Becton Dickinson, Sunnyvale, CA, USA) flow cytometer with excitation at 488 nm and emission at 525 nm.
The proportion of pyroptosis in NPCs was measured with the FAM FLICA™ Caspase-1 Kit (ICT098, Bio-Rad, CA, USA). After incubation with FAM FLICA (10 μl of 30x FLICA solution in 290 μl RPMI 1640) at 37°C for 30 min, the pyroptotic NPCs were analyzed using flow cytometry with excitation at 494 nm and emission at 520 nm.
The proportion of pyroptosis in NPCs was measured with the FAM FLICA™ Caspase-1 Kit (ICT098, Bio-Rad, CA, USA). After incubation with FAM FLICA (10 μl of 30x FLICA solution in 290 μl RPMI 1640) at 37°C for 30 min, the pyroptotic NPCs were analyzed using flow cytometry with excitation at 494 nm and emission at 520 nm.
4
Intracellular ROS Measurement using DCFH-DA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Production of intracellular ROS was measured using DCFH‐DA (Beyotime, Shanghai, China) in accordance with the manufacturer's instructions. In brief, the cells were trypsinized, incubated with 10 µmol/L DCFH‐DA at 37°C for 20 minutes, rinsed with RPMI‐1640 and analysed by flow cytometry (BD Biosciences, Franklin Lakes, NJ) at an excitation wavelength of 480 nm and emission wavelength of 525 nm.
5
Measurement of Intracellular ROS Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the intracellular ROS levels, MDA-MB-231 and MDA-MB-468 cells were pretreated with various concentrations of PP or 0.1% DMSO for 24 h and then incubated in the dark with 10 mM of the oxidation-sensitive fluorescent probe 2′7′-dichlorfluorescein-diacetate (DCFH-DA, Beyotime) for 20 min at 37°C. DCFH-DA was cleaved by intracellular esterase to liberate free DCFH, and the ROS level was measured using a BD Accuri C6 flow cytometer.
6
ROS Measurement using DCFH-DA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The intracellular ROS was measured using DCFH-DA (Sigma-Aldrich). This molecule diffuses through cell membranes and is hydrolyzed by intracellular esterase to liberate the free acid (2′, 7′ dichlorodihydrofluorescin (DCFH2)) which is trapped in the cells. DCFH2, upon reaction with oxidizing species, forms the highly fluorescent compound 2′, 7′- dichlorofluorescein (DCF). Thus, the fluorescence intensity was proportional to the amount of hydrogen peroxide produced by the cells. MDA-MB-231 cells were exposed to OEO/thymol for 12 h. After washing with PBS, the cells were incubated in DCFH-DA (20 μM) and kept in the dark for 15 min, and the intracellular fluorescence was measured using flow cytometry (FACSCalibur, BD Biosciences) with excitation and emission settings of 485–495 nm/525–530 nm, respectively81 (link),82 (link).
7
Apoptosis and ROS Detection Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell apoptosis was detected in MC-3T3-E1 cell cultures using a flow cytometer. The cells were harvested and re-suspended in Annexin binding buffer at 1×105 cells/ml. Then, the suspension was incubated with Annexin V-FITC and propidium iodide (PI) [cat. no. 70-AP101-60; MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China] in the dark for 15 min at 4°C. The apoptosis of the cell samples was analyzed by flow cytometry with BD CellQuest Pro Software version 1.2 (BD Biosciences, San Jose, CA, USA).
The ROS levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously described (39 (link)). DCFH-DA (Sigma-Aldrich; Merck KGaA), without fluorescence, can enter the cell membrane and form DCFH in the cell. DCFH is then oxidized to form a fluorescent substance DCF in the presence of ROS. MC-3T3-E1 cells were stained with DCFDA and held for 30 min at room temperature. Finally, DCF fluorescence levels were measured by flow cytometry and the data were analyzed using Summit Software (version 4.3; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA).
The ROS levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously described (39 (link)). DCFH-DA (Sigma-Aldrich; Merck KGaA), without fluorescence, can enter the cell membrane and form DCFH in the cell. DCFH is then oxidized to form a fluorescent substance DCF in the presence of ROS. MC-3T3-E1 cells were stained with DCFDA and held for 30 min at room temperature. Finally, DCF fluorescence levels were measured by flow cytometry and the data were analyzed using Summit Software (version 4.3; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA).
8
Intracellular ROS Quantification by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular reactive oxygen species (ROS) generation was assessed flow cytometry using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, Shanghai, China) following instructions. After collection, cells were incubated with the DCFH-DA dye, which was diluted in serum-free 1640 medium at a proportion of 1:1,000 for 30 min at 37°C in the dark, washed twice with PBS, and then resuspended in PBS to detect the generation of intracellular ROS by flow cytometry (BD C6 Biosciences). In all experiments, 10,000 viable cells were analyzed. Data analysis was performed by using Graph prism 8.0.
9
Measuring Oxidative Stress in GBM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 2–4 ×105 GBM cells were inoculated in each 6-well plate, and the cells were treated with diluted DMSO or 4 μM RB for 48 h. After 48 h of treatment, the cells were washed three times with PBS and incubated with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma–Aldrich) for 30 min. After incubation, DCFH-DA was replaced with PBS. The fluorescence intensity of DCFH-DA was detected by flow cytometry (BD C6, San Jose, CA, United States). FlowJo software was used to analyze flow cytometry data (Treestar; Ashland, OR, United States), and at least 1 × 105 cells were analyzed.
10
Measuring Cellular Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ROS generation was detected with 2′,7′-dichlorodihydrofluorescine diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA). Briefly, cells cultured in 35-mm-diameter glass-bottom culture dishes (MatTek, Ashland, MA, USA) were incubated with 10 μM DCFH-DA for 10 min at 37°C in serum-free DMEM, then washed twice with Dulbecco’s phosphate-buffered saline, and analyzed using FACSCalibur flow cytometer (BD Biosciences). The mean fluorescence intensity was analyzed using the CellQuest software version 5.2 (BD Biosciences).
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!