After 48 hours of transfection, the cells were seeded in 6-well plates. After 8 h, the cells were stained with 20 μM 2’,7’-dichlorofluorescein diacetates (Beyotime Biotechnology, Shanghai, China) in the dark at 37°C for 30 min. The ROS level in the cells was observed by a fluorescence microscope (28 ).
2 7 dichlorofluorescein diacetate
Manufactured by Beyotime
Sourced in China
2′,7′-dichlorofluorescein diacetate is a fluorescent dye used as a reagent in various cell-based assays. It is a non-fluorescent compound that is converted to a highly fluorescent product upon hydrolysis by intracellular esterases. The fluorescent product can be detected using appropriate excitation and emission wavelengths, making it useful for monitoring cellular processes and viability.
Automatically generated - may contain errors
Lab products found in correlation
Jc 1 dye solution, by Beyotime (4 mentions)
Rnase a, by Beyotime (3 mentions)
Facscalibur, by BD (3 mentions)
Pi annexin 5 solution, by Keygen Biotech (3 mentions)
Flow cytometry, by Beckman Coulter (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Rhodamine 123, by Beyotime (2 mentions)
Piperlongumine, by Energy Chemical (2 mentions)
Thiobarbituric acid, by Beyotime (2 mentions)
Substituted benzaldehyde, by Energy Chemical (2 mentions)
2 piperidone, by Energy Chemical (2 mentions)
Gsh and gssg assay kit, by Beyotime (2 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (2 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Mda assay kit, by Beyotime (1 mentions)
Gsh assay kit, by Beyotime (1 mentions)
Jc 1 staining solution, by Beyotime (1 mentions)
Hoechst 33342 staining, by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
24 protocols using 2 7 dichlorofluorescein diacetate
1
Oxidative Stress Measurement Protocol
2
Cerebral Cortex Homogenate Analysis
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In vivo, approximately 0.2–1 g of cerebral cortex in control and model rats were isolated, washed with saline at 4 °C, cut into pieces, and grinded into homogenate. The prepared homogenate was centrifuged at 3,000 × g for 10–15 min under low temperature.
In vitro, the concentrations of Fe ion, GSH, ROS, GPX4, and malondialdehyde (MDA) in groups of iron-rich, control, model, and model + miR-9-5p angomir, Vector, WT NEAT1, and MUT NEAT1 were measured. Cells were placed at room temperature for 1 h, centrifuged at 1,000–2,000 × g at 4 °C for 10 min before the supernatant was collected. Then, a commercial iron assay kit, GSH assay kit, GPX4 assay kit, and MDA assay kit, purchased from Beyotime Biotechnology (Shanghai, China) were used for the level evaluation of Fe iron, GSH, GPX4, and MDA, respectively. The ROS level was measured by employing the 2′,7′-dichlorofluorescein diacetates (Beyotime Biotechnology, Shanghai, China). The cells were placed at 37 °C for 30 min in the dark, harvested with 0.05% trypsin–EDTA solution, suspended in a fresh medium, and finally photographed and observed by a fluorescence microscope (Leica) [31 (link)]. Each experiment was tripled.
In vitro, the concentrations of Fe ion, GSH, ROS, GPX4, and malondialdehyde (MDA) in groups of iron-rich, control, model, and model + miR-9-5p angomir, Vector, WT NEAT1, and MUT NEAT1 were measured. Cells were placed at room temperature for 1 h, centrifuged at 1,000–2,000 × g at 4 °C for 10 min before the supernatant was collected. Then, a commercial iron assay kit, GSH assay kit, GPX4 assay kit, and MDA assay kit, purchased from Beyotime Biotechnology (Shanghai, China) were used for the level evaluation of Fe iron, GSH, GPX4, and MDA, respectively. The ROS level was measured by employing the 2′,7′-dichlorofluorescein diacetates (Beyotime Biotechnology, Shanghai, China). The cells were placed at 37 °C for 30 min in the dark, harvested with 0.05% trypsin–EDTA solution, suspended in a fresh medium, and finally photographed and observed by a fluorescence microscope (Leica) [31 (link)]. Each experiment was tripled.
3
ROS Measurement in Treated RPE Cells
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The treated RPE cells were incubated with 10 μM of 2′-7′-dichlorofluorescein diacetate (Beyotime, Shanghai, China) for 30 min at 37 °C and washed with PBS. The ROS secreted by RPE cells promoted the production of 2′-7′-dichlorofluorescein which is a fluorescent compound. The intracellular fluorescence was observed by a Fluorescent Microscope (DP73, Olympus, Tokyo, Japan).
4
Measuring Intracellular ROS in RAW264.7 Cells
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The macrophage cell line (RAW264.7) was used for the analysis of ROS production. Cells were cultured in DMEM medium (Cat#12800-017; Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (Cat#2177370; Gibco, Grand Island, NY, USA) in an incubator at 37 °C, 5% CO2 in the humid atmosphere. Cultured cells were divided into three groups: (1) vehicle: same volume solvent was added; (2) UII group: 100 nM UII for 24 h; (3) UII + Urantide group: cells were co-treated with 100 nM UII plus 100 nM urantide for 24 h. After treatment with UII or urantide, the cells in the 12-well culture dishes were loaded with 10 μM of 2′,7′-dichlorofluorescein diacetate (Cat#S0033; Beyotime, Shanghai, China) and cultured for another 0.5 h. Cultured cells were observed under a fluorescent inverted microscope in a dark room. ROS levels were quantified as the mean fluorescence intensity by image software.
5
Cell Cycle and Mitochondrial Assay
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Cells were inoculated in 6-well plates and processed according to the protocol described in Section 2.2 . Cells were digested with trypsin and washed twice with pre-chilled phosphate-buffered saline (PBS), before being maintained at 4 °C overnight with 75% ethanol. Cells were washed three times with pre-chilled PBS and maintained at 37 °C for 30 min with propidium staining and RNase A (Beyotime, Shanghai, China), and a Flow Cytometer (Beckman Coulter, Brea, CA, USA) was used to analyze the cell cycle. To evaluate the reactive oxygen species (ROS) production, cells were digested and washed once in pre-chilled PBS, then maintained at 37 °C for 20 min with 10 μM of 2,7-dichlorofluorescein diacetate (Beyotime, Shanghai, China). The cells were washed three times and the ROS level was tested with a flow cytometer. Cells were digested and maintained at 37 °C for 20 min in the dark with 1 mL growth medium and 1 mL 1 × JC-1 staining solution (Beyotime, Shanghai, China). Then, the cells were washed with pre-chilled PBS and cultured in 1× JC-1 staining buffer. The mitochondrial membrane potential (∆Ψm) of the cells was detected utilizing flow cytometry.
6
Cellular Imaging with Fluorescent Probes
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Hoechst 33342 Staining and 2,7-dichlorofluorescein diacetate (Beyotime Biotechnology) working solutions were added to cells. After incubation for 20 min at 37 °C, the cells were washed three times. The images were scanned under a confocal laser-scanning microscope (SP8; Leica).
7
Apoptosis and Oxidative Stress Evaluation
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Reagents were freshly diluted to the desired concentration with the culture medium before use. Fetal bovine serum (FBS), Roswell Park Memorial Institute 1640 (RPMI 1640), Dulbecco's modified Eagle's medium (DMEM), penicillin (100 U/ml), and streptomycin (100 µg/ml) were purchased from Gibco (Thermo Fisher Scientific, Inc.). The Annexin V-FITC Apoptosis Detection Kit, Cell Cycle Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and N-acetyl-L-cysteine (NAC) were purchased from Beyotime Institute of Biotechnology. DMSO was obtained from Sigma-Aldrich (Merck KGaA). All primary antibodies used in the present study were purchased from Santa Cruz Biotechnology, Inc. and all secondary antibodies used were purchased from ZSGB-BIO.
8
ROS Detection by Flow Cytometry
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Cells were harvested and stained with H2O2-sensitive dye (5 μM), 2',7'-dichlorofluorescein-diacetate (Beyotime Institute of BioTechnology) and · O2−-sensitive dye dihydroethidium (Beyotime Institute of BioTechnology). The fluorescence intensity was quantified by FACSCalibur flow cytometry (Becton Dickinson) at Ex./Em.=488 nm / 525 nm for H2O2 detection and Ex/Em=300 nm/610 nm for · O2− detection, respectively.
9
Intracellular ROS Detection with DCFDA
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The peroxide sensitive fluorescent probe 2′7’-dichlorofluorescein diacetate (Beyotime Institute of Biotechnology) was used to detect intracellular ROS according to a previous study [20 (link)]. After treatment as indicated, MAC-T cells were incubated with 25 μmol/L 2′,7'-dichlorofluorescein diacetate in serum-free DMEM/F12 medium at 37 °C for 20 min. Cells were then washed twice with PBS and resuspended with serum-free DMEM/F12 medium. The fluorescence was analyzed by flow cytometry (FACSCalibur, Becton-Dickinson, Sunnyvale, CA, USA). Results were expressed as fold changes by normalizing the data to the control values.
10
Intracellular ROS Measurement in KGN Cells
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Intracellular ROS levels were evaluated with 2′,7′-dichlorofluorescein-diacetate (Beyotime, China) according to the manufacturer's guidelines. In summary, KGN cells from each group were cultured in 48-well plates and then incubated with 500 μL of 75 μM H2DCF-DA for 20 min in the dark at 37 °C. The incubated cells were subsequently washed three times with serum-free medium. Hoechst (Beyotime, China) stain was generated as a working solution and added to cells that had been treated as indicated. After incubation at 37 °C for 5 min, the cells were washed three times with serum-free medium, and images were taken with an IX71 microscope (Olympus, USA) in three randomly selected fields.
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