A DCFH-DA detection kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to assess the ROS level in HUVEC. The cells were seeded in 6-well plates, treated with the various samples, and incubated for 24 h. After washing the cells with the reduced-serum medium, 10 μmol/L DCFH-DA was added to each well, and the cells were incubated at 37°C for 20 min. The cells were then washed thoroughly with reduced-serum medium to remove the DCFH-DA that did not enter the cells. The cells were collected in 1 mL of PBS after dissociation, and the fluorescence was immediately recorded using a LB 941 TriStar Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) using 485 nm excitation and 535 nm emission filters. The total fluorescence intensity of the cells in each well was recorded, and ROS generation was measured as the fold increase over the untreated control.
Dcfh da detection kit
Manufactured by Beyotime
Sourced in China
The DCFH-DA detection kit is a laboratory reagent used to measure the levels of reactive oxygen species (ROS) in biological samples. It contains the chemical compound DCFH-DA, which is a fluorogenic probe that reacts with ROS to produce a fluorescent signal. The kit provides a simple and reliable method for researchers to quantify the presence and levels of ROS in their experiments.
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Lab products found in correlation
6 protocols using dcfh da detection kit
1
ROS Quantification in HUVECs
2
ARPE-19 ROS Generation Assay
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ARPE-19 cells were treated with adv-NC or adv-HTRA1 for 24–48 h, DCFH-DA detection kit (Beyotime) was used to determine ROS generation. Experiments were done according to the manufacturer’s instruction, the DCFH-DA fluorescence data were measured using flow cytometry.
3
ROS Modulation by Paclitaxel and NAC
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CHMm cells were treated with (0, 0.01, 0.1 and 1 µM) paclitaxel, with or without 5 mM N-acetyl-L-cysteine (NAC). After a treatment period of 24 h, cells were digested with 0.25% trypsin. Levels of ROS were measured using a dichlorodihydrofluorescein diacetate (DCFH-DA) detection kit, according to the manufacturer's protocol (Beyotime Institute of Biotechnology). Briefly, cells were collected (1×106/ml), washed with serum free DMEM and incubated with 10 mM DCFH-DA at 37°C for 20 min. Stained cells were washed three times and re-suspended in serum-free DMEM. Intracellular ROS oxidized the non-fluorescent DCFH resulting in conversion to the fluorescent DCF molecule. The levels of ROS were measured using a fluorescent microplate reader using a reference wavelength of 490 nm and a detection wavelength of 570 nm.
4
Measuring Oxidative Stress in HSC-T6 Cells
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The HSC‐T6 cell's ROS levels were measured by DCFH‐DA detection kit (Beyotime). Briefly, after Mv treatment (final concentrations: 0, 50, 75, and 100 μg/ml, respectively) for 24 hr, HSC‐T6 cells were stained with 10 μM/ml of H2DCFDA at 37°C for 20 min in the dark. The relative ROS levels in the HSC‐T6 cells were observed by an IX53 Inverted Fluorescent Microscope (Olympus). Fluorescent intensity was measured at the 530 nm emission wavelength and 485 nm excitation wavelength, respectively.
5
Intracellular ROS Measurement in HepG2 Cells
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The intracellular ROS production was measured by a DCFH-DA detection kit (Beyotime Biotechnology, China). The HepG2 cells were incubated with or without NIE for 6 h, incubated in the dark at 37°C, and kept away from light for 20 min with 10 μM DCFH-DA. PTEN inhibitor (Oroxin B) was used as the positive control. The cells were washed and then detected with a fluorescence microscope.
6
Quantifying Cellular ROS Levels
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The ROS levels in HK-2 cells were detected with the DCFH-DA Detection Kit (S0033, Beyotime, Shanghai, China). Briefly, the HK-2 cells were seeded in a 6-well plate and incubated with cisplatin or doxorubicin in the presence or absence of NAC for 48 h. After washing, the cells were stained with 10 μM of DCFH-DA at 37 °C for 30 min according to the manufacturer’s instructions.
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