The DCFH-DA staining was used to detect the endogenous ROS generation as described previously [19 (link)]. Briefly, after being treated with 50 μg/mL 18 for 48 h, R. solani mycelia tips (0.6 cm) were cut and then placed on a sterile slide in a 9-cm culture dish before continued incubation at 25 °C for 24 h. The PDA medium was removed, and the hyphae were stained with 10 μM DCFH-DA solution (Beyotime, Shanghai, China) and incubated at 37 °C for 30 min in the darkness. The samples were observed using a fluorescence microscopy (Nikon ECLIPES 80 i, Tokyo, Japan).
Dcfh da solution
Manufactured by Beyotime
Sourced in China
DCFH-DA solution is a chemical reagent used in cell-based assays. It is a non-fluorescent compound that becomes fluorescent upon oxidation, allowing the detection of reactive oxygen species (ROS) in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Eclipes 80 i, by Nikon (2 mentions)
Af6000, by Leica (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Fluoview v2, by Olympus (1 mentions)
Dulbecco modified eagle medium (dmem), by Beyotime (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Catalase assay, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
10 protocols using dcfh da solution
1
ROS Detection in R. solani Mycelia
2
Assaying ROS in R. solani Hyphae
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The R. solani hyphae were treated with 0 and 1 μg/mL compound 55 for 48 h, and then were cut and placed on a sterile slide before continued incubation at 25 °C for 24 h. The hyphae were stained with 10 μM DCFH-DA solution (Beyotime, Shanghai, China). After incubation at 37 °C for 30 min in the darkness, the samples were observed and photographed using a fluorescence microscopy (Nikon ECLIPES 80 i, Tokyo, Japan).
3
Quantifying Intracellular Reactive Oxygen Levels
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Cells were immersed in 10 μmol/L DCFH-DA solution (S0033S, Beyotime Biotechnology, Shanghai, China) for 30 min, and then washed with a serum-free medium 3 times. The degree of ROS level was detected using a laser scanning confocal microscope (AF6000, Leica, Wetzlar, Germany). Using Image J software (v1.8.0.112), the image threshold was adjusted, ROS signal region was selected, and mean fluorescence intensity values of the signal region was quantified after setting the measured parameters.
4
Intracellular ROS Detection Using DCFH-DA
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Intracellular ROS level was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA) solution (Beyotime Institute of biotechnology, Wuhan, China). DCFH-DA was diluted with a serum-free medium according to the manufacturer’s instructions. The cells were collected and re-suspended in the DCFH-DA working solution with a density of 1 × 106-2 × 107 cells/ml, and incubated for 20 min at 37°C. Rosup reactive oxygen stimulant was added to the positive control cell suspension as the positive control. Then, the cells in each group were filtered through a 200-mesh filter, and a flow cytometer was used to detect the fluorescence intensity of DCF at the excitation wavelength of 488 nm.
5
Intracellular ROS Measurement by DCFH-DA
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The ROS probe DCFH-DA was used to investigate the intracellular ROS level via CLSM and FCM analysis. Briefly, CT26 cells were seeded into 6-well microplates (106 cells for each well) for 24 h (37 °C, 5% CO2). Twenty-four hours following incubation with BST NSs, CaO2 NPs and BST/CaO2 NSs, CT26 cells were washed and incubated with 0.2 μM DCFH-DA solution (S0033; Beyotime) for 1 h at 37 °C. After rinsing with dilution buffer, the intracellular ROS level was measured using CLSM and FCM analysis according to the manufacturer’s protocol. Analysis of FCM data was performed with Flowjo v7.6 software.
6
ROS Detection in H1299 Cells
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After the H1299 cells adhered in confocal chamber slides, HmN, HmDN, AHmDN or HHmDN was added into the medium with the final concentration of mPPZ 0.5µM. After the incubation for 12hrs, cells were washed by PBS and then incubated with 10μmol/l 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) solution (S0033, Beyotime, China) for 30mins in serum-free medium in the dark. The illumination (680nm) at a light fluence of 1.5 J/cm2 was followed by ROS Assessment. The fluorescence, based on the oxidative conversion of DCFH-DA to dichlorofluorescein (DCF) by ROS, was detected using Olympus FluoViewTM FV1000 laser scanning confocal microscope (Olympus Corp., Tokyo, Japan) with the excitation at 488 nm. All images were analysed by Olympus Fluoview v2.1 software.
7
Intracellular ROS Quantification in A375 Cells
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As reported previously [21 (link), 22 (link)], the A375 cells (3 × 105 cells/well) were seeded in 6-well plates and cultured for 24 h, which were further treated by EU-5 (0, 2.2 and 4.4 µM) for 48 h. Then the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) solution (10 µM, Beyotime) was added into the treated cells, which were incubated at 37 oC for 20 min. After washing and digestion, the cells were collected and detected by flow cytometer. The data was analyzed by FlowJo VX software.
8
Evaluating PMPB Nanocomposite's Antioxidant Potential
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Four groups, i.e., control, model, PMPB (50 μg/mL) and PMPB (100 μg/mL), were set to explore the ROS-scavenging capability of PMPB NC. RAW264.7 cells were cultured in 12-well plates for 12 h. Except control group, cells in other groups were stimulated with LPS (100 ng/mL) and IFN-γ (100 IU/mL). The cells in PMPB NC group were pretreated with PMPB NC for 2 h, and then stimulated with LPS/IFN-γ for 4 h. Those cells in control group were treated with fresh medium, and the cells in model group were stimulated with LPS/IFN-γ without treatment. Subsequently, the culture media were replaced by DCFH-DA solution (100 μ 1/9 μl in DMEM, Beyotime Biotechnology). Cells were rinsed and treated with DCFH-DA (10 μM) in serum-free DMEM. After another 30 min, the cells were finally rinsed with PBS for three times and then observed by CLSM.
9
Intracellular ROS Quantification
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Intracellular ROS level were measured with a 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) solution (Beyotime, Shanghai, China) according to the manufacturer's instructions. Hydrogen peroxide served as a positive control. Image-Pro Plus software was used to quantitatively detect the intracellular fluorescence indicative of ROS.
10
Oxidative Stress Response in C. elegans
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With reference to Song et al (6 (link)), CL4176 nematodes were synchronized at 16˚C for 48 h, cultured at 25˚C for 40 h then rinsed twice with M9 buffer to remove E. coli. The nematodes were then homogenized and protein abundance was measured by the bicinchoninic acid assay (Beyotime Institute of Biotechnology). The levels of MDA, SOD and CAT were determined using Total Superoxide Dismutase Assay (Beyotime Institute of Biotechnology), Catalase Assay (Beyotime Institute of Biotechnology) and Malondialdehyde Detection (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. ROS accumulation was determined with reference to Wang et al (29 (link)). Briefly, 50 µl of nematode supernatant was added to each well of a 96-well plate and 50 µl of 100 µM DCFH-DA solution (a fluorescent probe) (Beyotime Institute of Biotechnology) was added to give a final DCFH-DA concentration of 50 µM, The solutions were thoroughly mixed by shaking for 30 sec. Fluorescence detection was performed using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Detection was conducted once every 10 min, and ROS changes within 100 min were counted.
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