ROS levels in the oocytes were assessed in accordance with the manufacturer’s procedure [35 (link)]. MII oocytes subjected to different experimental conditions were incubated in α-MEM supplemented with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (Beyotime Institute of Biotechnology) at 37 °C for 30 min, then washed with PBS at least three times. Images of the oocytes were acquired using a fluorescence microscope using the same settings. Fluorescence intensity was calculated using ImageJ software.
2 7 dichlorodihydrofluorescein diacetate
Manufactured by Beyotime
Sourced in China
2',7'-dichlorodihydrofluorescein diacetate is a fluorogenic probe used for the detection and measurement of oxidative stress in biological systems. It is a non-fluorescent compound that becomes highly fluorescent upon oxidation, allowing for the quantification of reactive oxygen species.
Automatically generated - may contain errors
Lab products found in correlation
α mem, by Beyotime (1 mentions)
Dulbecco s modified eagle medium dmem, by Solarbio (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Maclin Biochemical Technology (1 mentions)
Tris 2 dimethylamino ethyl amine, by Merck Group (1 mentions)
N butyl acrylate, by Maclin Biochemical Technology (1 mentions)
Chlorin e6 ce6, by Maclin Biochemical Technology (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Trypsin, by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Fluorescence microplate reader, by Tecan (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facsuite, by BD (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Atp determination kit, by Beyotime (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
12 protocols using 2 7 dichlorodihydrofluorescein diacetate
1
Measuring Oocyte ROS Levels
2
ROS Detection in HL-1 Cells
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The fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate (Beyotime Institute of Biotechnology) was used to detect reactive oxygen species (ROS) levels, the detected method as described in our previous study (Xu C. et al., 2018). The HL-1 cells (2 × 104 per well) were seeded into 24-well plates, then incubated with PA (0.25 mM) for 24 h to induce ROS generation. CAN (2.5 μg/ml) was simultaneously treated for 24 h to investigate the activity against ROS. The blank and untreated control cells were treated with an equal volume of DMSO or BSA solution. The fluorescent signal was measured using a fluorescence microscope (excitation wavelength of 485 nm; emission wavelength of 525 nm). Finally, quantitative analysis was conducted by ImageJ.
3
Photodynamic Therapy Nanoparticle Synthesis
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Poly(ethylene glycol) methyl ether acrylate (PEGA), Ethyl 2-bromoisobutyrate (EBIB), and Tris [2-(dimethylamino) ethyl] amine were purchased from Sigma-Aldrich (Shanghai, China). n-Butyl acrylate and Isopropyl alcohol (IPA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Chlorin e6(Ce6), and Perfluoro-15-crown-5-ether (PFCE) were obtained from Macklin (Shanghai, China). Fetal bovine serum (FBS) was purchased from Every Green (Hangzhou, China). Dulbecco’s Modified Eagle Medium (DMEM), Phosphate-Buffered Saline (PBS), and Trypsin were purchased from Solarbio (Beijing, China). 2′,7′-Dichlorodihydrofluorescein diacetate, Singlet Oxygen Sensor Green, and Hoechst 33342 were obtained from Beyotime (Shanghai, China). All of the chemicals were used as supplied without further purification.
4
Cellular ROS Visualization via DCFDA
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Cells were seeded on six-well plates and cultured with 10-μM 2′,7′-dichlorodihydrofluorescein diacetate (Beyotime, Wuhan, China) for 30 min at 37°C, avoiding darkness. After three washes with ice-cold PBS, cells were imaged using a fluorescence microscope at 488 nm excitation and 525 nm emission wavelengths.
5
Intracellular Reactive Oxygen Assay
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Cells were cultured at a density of 1.8x105 cells/ml, and then incubated for 24 h. The cells with different treatments were then treated with 2',7'-dichlorodihydrofluorescein diacetate (10 µM; Beyotime Institute of Biotechnology) in the dark, the cells were resuspended in PBS, followed by an examination of the fluorescence intensity using a microscope. Image J was applied for quantification.
6
ROS Detection in Cell Cultures
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The cells were collected and incubated with 2′, 7′-dichlorodihydrofluorescein diacetate (Beyotime) for 30 min, and ROS activity was detected using a fluorescence microscope (Nikon) or fluorescence microplate reader (Tecan) with excitation at 488 nm and emission at 525 nm.
7
Measurement of Intracellular ROS Levels
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Cells were seeded into six-well plates at an appropriate density (1 × 105 cells/well) for 24 h, and then treated with Pur or DDP alone or in combination as mentioned above for 24 h. The amount of ROS produced in cells was measured by 2′,7′-dichlorodihydrofluorescein diacetate (Beyotime Biotech, Nantong, China) according to the manufacturer’s instructions. Cells were stained with 10 μM DCFH-DA for 30 min at 37°C. Then, the cells were collected and washed twice with pre-chilled PBS. The fluorescence intensity was analyzed using a FACS Calibur flow cytometer (BD Biosciences, California, USA). To assess the changes in ROS after using inhibitors, cells were pretreated with 5 mM NAC for 2 h prior to exposure to compounds.
8
Cellular ROS Detection in HK-2 Cells
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HK-2 cells were cultured in 6-well confocal plates (2 × 105 cells per well). The cells were treated with 10 μM cisplatin and 50 μM CNPs, then co-incubated for 24 h. Cellular ROS was detected using a fluorescence probe (2,7-dichlorodihydrofluorescein diacetate, Beyotime Biotechnology, China) under the fluorescence microscope. In addition, ROS were also detected by the FITC channel for fluorescence intensity using flow cytometry (BD Biosciences, FACSuiteTM, USA).
9
ROS Quantification by DCFH-DA
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Cellular ROS levels were measured by 2′,7′-dichlorodihydrofluorescein diacetate (Beyotime).
10
Corilagin Modulates Cellular ROS in Osteoclasts
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Cellular reactive oxygen species (ROS) levels play an important role in osteoclast differentiation. To determine whether ROS levels changed during Corilagin treatment, RAW264.7 cells were pre‐treated with or without Corilagin for 24 hours. Then, cells were cultured with 10 mmol/L 2′,7′‐dichlorodihydro‐fluorescein diacetate (Beyotime Biotechnology) for 20 minutes in the dark. After that, cells were stimulated with 100 ng/mL RANKL for 30 minutes, and the intracellular dichlorofluorescein intensity was analysed using a multimodal microplate reader (SpectraMaxM5; Molecular Devices).
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