The primary peritoneal macrophages (2 × 106 cells/well) were seeded into the 6-well plates. Dilute DCFH-DA (Nanjing Jiancheng Bioengineering Institute, China) with RPMI 1640 at the ratio of 1:1000 to a final concentration of 10 µM. Add 1 mL diluted DCFH-DA solution to each well and incubate for 20 min at 37 ℃. The cell suspension was obtained by adding 1 mL RPMI 1640, then centrifuged at 1500 rpm for 5 min, followed by resuspension with 400 µL PBS for flow cytometry assay (BD, USA).
Dcfh da
Manufactured by Nanjing Jiancheng
Sourced in China, United States
DCFH-DA is a fluorescent probe commonly used in cellular assays. It is a cell-permeable dye that can be used to detect the presence of reactive oxygen species (ROS) within cells. Upon oxidation, DCFH-DA emits a green fluorescent signal that can be measured using a fluorescence plate reader or microscope.
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Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Facscalibur, by BD (3 mentions)
Confocal laser scanning microscope, by Leica (2 mentions)
Microplate reader, by Tecan (2 mentions)
Fc500, by Beckman Coulter (2 mentions)
Fluorescence microscopy, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dcfh da, by Olympus (2 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Cellquest program, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
E004 1 1, by Nanjing Jiancheng (1 mentions)
Cd44 apc, by BioLegend (1 mentions)
Cd90 fitc, by BioLegend (1 mentions)
Cd34 pe, by BioLegend (1 mentions)
Flow cytometry, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Dcfh da, by BD (1 mentions)
97 protocols using dcfh da
1
Quantifying Macrophage Oxidative Stress
2
Fluorescent Oxidative Stress Assay
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Dilute DCFH-DA (Nanjing Jiancheng Bioengineering Institute, China) with DMEM/F12 at the ratio of 1:1000 to a final concentration of 10 μM. Add 1 mL diluted DCFH-DA solution to each well and incubate for 20–30 min at 37 °C. The MLE12 cells were washed three times with PBS and photographed with fluorescence microscopy (Nikon, Japan).
3
Measuring Cellular Oxidative Stress Response
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Cell suspensions were incubated at 37 °C for 30 min with 10 µM 2ˈ,7ˈ dichlorofluorescein diacetate (DCFH/DA, Nanjing Jiancheng Bioengineering Institute, China). To measure the intracellular reactive oxygen species (ROS), the fluorescence of DCF was excited by a 15mW laser tuned to 488 nm and the emitted fluorescence was measured with 530/30 band pass filter in a FACScalibur Becton Dickinson flow cytometer. The conditions for data acquisition and data analysis were established using negative and positive controls with the CellQuest Program of Becton Dickinson and these conditions were maintained during all the experiments. Total superoxide dismutase (SOD) activity was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China) according to Nakano (1990) . Glutathione peroxidase (GPX) activity was measured using the method of Dabas et al. (2012) . Thiobarbituricacid-reactive substances assays were performed with amalondialdehyde (MDA) kit (Nanjing Jiancheng Bioengineering Institute, China) as described by Rueda-Jasso et al.
(2004).
(2004).
4
ROS Production Measurement in Cells
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The ROS production in cells was determined by a commercial kit (Nanjing Jiancheng, E004-1-1). As described in the manufacture’s protocol, the cells of each sample were incubated at 37 °C for 1 h in the serum free medium containing DCFH-DA (1:1000) (Nanjing Jiancheng, E004-1-1). Then the cells were digested by trypsin (Gibco), washed by PBS for 2–3 times, and suspended in PBS. The fluorescence signals were detected at the excitation wavelength 485 nm and the emission wavelength 525 nm (Synergy HTX multifunction).
5
Hepatocyte ROS Measurement Using DCFH-DA
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2,7-Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used to measure the ROS content in the hepatocytes following the kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Approximately 1 mm3 liver tissue sample was taken and washed with PBS for subsequent digestion with trypsin enzyme at 37°C. The solution was screened using a nylon mesh, washed twice with PBS, and centrifuged to get a single-cell suspension. Later, DCFH-DA was added with dimethyl sulfoxide (DMSO) as solvent. The sample was incubated at 37° C for 1 hour in the dark, and fluorescence was observed at an optical excitation wavelength of 485 nm and an optical emission wavelength of 510 nm using a spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland).
6
Multiparametric Analysis of Porcine Mesenchymal Stem Cells
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To identify cellular surface immunophenotypes, porcine MSCs were digested and washed twice with PBS. The cells were labeled with antibodies against PerCP-CD45 (Cat.#: 642275; BD Biosciences, New York, NY, USA), APC-CD44 (Cat.#: 103011; BioLegend, San Diego, CA, USA), FITC-CD90 (Cat.#: 328107; BioLegend, CA, USA), and PE-CD34 (Cat.#: 343605; BioLegend, CA, USA) for 30 min. After washing twice with PBS, the labeled cells were analyzed using a flow cytometer (BD Biosciences, New York, NY, USA).
To measure the intracellular reactive oxygen species (ROS) level, cells were incubated with 10 μM of DCFH-DA (Nanjing Jiancheng Biotechnology, Nanjing, China) at 37 °C for 1 h, followed by washing and resuspending with PBS. Fluorescence was analyzed via flow cytometry (BD Biosciences, New York, NY, USA) with excitation at 500 nm and emission at 525 nm.
To measure the intracellular reactive oxygen species (ROS) level, cells were incubated with 10 μM of DCFH-DA (Nanjing Jiancheng Biotechnology, Nanjing, China) at 37 °C for 1 h, followed by washing and resuspending with PBS. Fluorescence was analyzed via flow cytometry (BD Biosciences, New York, NY, USA) with excitation at 500 nm and emission at 525 nm.
7
Kidney Oxidative Stress and Inflammation Assessment
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Urine was collected before the mice were sacrificed to determine urine protein. The blood sample was centrifuged at 3,000 rpm/min to obtain serum. Serum creatinine (Cr) and blood urea nitrogen (BUN) detections were performed to evaluate kidney function following protocols. For reactive oxygen species (ROS) detection, HK-2 cells and kidney tissues were stained with a 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence probe using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The oxidative indices were measured in kidney tissues to assess renal oxidant injury, including glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and malondialdehyde (MDA). ELISA Kits of IL-6, IL-1β, and TNF-α were utilized to quantity inflammatory cytokine concentration in both serum and kidney tissues according to the manufacturer’s instructions.
8
Cytotoxicity and ROS Evaluation
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Briefly, cells were seeded in a 96-well microplate (1 × 104 cells/well) and were cultured for 24 h. After treated with rotenone, MPP+ and irisin as previously described, the medium was replaced by CCK8 (DOJINDO, China, CK04, diluted 10 times in medium to use) or DCFH-DA (Jiancheng, China, E004, final concentration 10μm), followed by incubation at 37 °C for 4 h. Then the CCK8 level and DCFH-DA level were detected by automated microplate reader (SpectraMax iD3).
9
Measuring Intracellular ROS Generation
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Intracellular reactive oxygen species (ROS) generation was measured with diacetylated 2′,7′-dichlorofluorescein (DCFH-DA, Nanjing Jiancheng Bioengineering Institute, China). In brief, 106 cells were incubated in 60 mm plates and, 24 h later, treated with various compounds, as indicated in the legends to figures. Cells were incubated with 10 μM of DCFH-DA for 20 min at 37°C, and the DCF fluorescence was measured by flow cytometry (Becton Dickinson FACScan, USA) as previously described in detail [18 (link)]. Data were analyzed using Cellquest software (Becton Dickinson). All data are presented as the mean of three independent experiments.
10
Oxidative Stress Evaluation using DCFH-DA
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The cells were incubated with 2,7-dichlorofluorescein-diacetate (DCFH-DA, 50 ng/mL, Jiancheng, China) at 37°C for 30 min in the dark. The cells were subsequently washed with PBS and observed under a confocal laser scanning microscope (Leica), and the images were captured. The mean fluorescence intensity of dichlorofluorescein (DCF) was analyzed.
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