Mitochondrial membrane potential in rat brain was detected by mitochondrial membrane potential assay kit with JC-1 (C2006, Beyotime) as described previously (C2006, Beyotime) (Ma et al., 2010). After brain tissue mitochondria extraction, JC-1 dyeing solution diluted five times with the JC-1 dyeing buffer. We added 0.1 mL of the purified mitochondria (total protein content, approximately 10–100 µg) to 0.9 mL of the diluted JC-1 staining solution and mixed the sample. A fluorescence spectrophotometer (Thermo Fisher Scientific) was used for analysis (excitation wavelength, 485 nm, emission wavelength, 590 nm).
Fluorescence spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Fluorescence Spectrophotometer is a scientific instrument used to measure the fluorescence properties of a sample. It excites the sample with light at a specific wavelength and detects the emitted light at different wavelengths, providing information about the fluorescence characteristics of the sample.
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Lab products found in correlation
Dcfh da, by Beyotime (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Dibac4 3, by Thermo Fisher Scientific (1 mentions)
Dharmafect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Gus staining kit, by Solarbio (1 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (1 mentions)
Prism 5, by GraphPad (1 mentions)
Miseq platform, by Illumina (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Dx 4000 fitc, by Merck Group (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Avance 500 spectrometer, by Bruker (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe, by Beyotime (1 mentions)
Jc 1 probe, by Beyotime (1 mentions)
A1r mp, by Nikon (1 mentions)
29 protocols using fluorescence spectrophotometer
1
Mitochondrial Membrane Potential Assay
2
Quantifying NP Cell DNA in Scaffolds
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The total DNA content of NP cells in the cell-seeded scaffolds was determined by fluorescence spectrophotometer (Thermo Fisher Scientific, USA). The assays were performed at day 4, week 2 and week 4, respectively. Cells cultured in wells without scaffold at a density of 2 × 107 cells/cm2 were served as controls. The cell-seeded scaffolds were washed with PBS (Sigma, St. Louis, MO, USA), crushed and air-dried under the laminar airflow for 10 min and frozen at −50 °C for 24 h. The scaffolds were then lysed with Nuelei Lysis (Promega, USA) solution for 30 min at room temperature. Afterwards, the lysed product was added to 100 μl DNA rehydration working reagent (A1125, Wizard genomic DNA purification kit, Promega, USA) and incubated at 37 °C. The absorbance of these samples was measured at 37 °C with a 458 nm fluorescence spectrophotometer. The six samples in each group were measured at each time point and the results were recorded in U L–1. Morphological changes of the cell-seeded scaffolds cultured in the induced medium were observed at every time point in vitro. The pure scaffold without seeded cells and the normal NP were used as the negative and positive control, respectively.
3
Fluorescence-Based Membrane Potential Assay
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Cell membrane depolarization was measured using the membrane potential–sensitive dye bis-(1,3-dibutylbarbituric acid) trimethin eoxonol [DiBAC4(3), Invitrogen]. Upon transmembrane depolarization, the DiBAC4(3) enters the cell and binds to protein molecules, acquiring enhanced fluorescence. By contrast, fluorescence intensity is reduced when the membrane is hyperpolarized. As previously described7 (link),73 (link), after incubation with 0.5 µM DiBAC4(3) for 30 minutes at 37 °C, cells were subjected to time scanning using a fluorescence spectrophotometer (Thermo Scientific) with excitation at 488 nm and emission measured at 518 nm. Membrane depolarization was monitored by observing changes in the intensity of fluorescence emission of DiBAC4(3). All values of fluorescence image analyses were corrected for cell number and background fluorescence.
4
Quantitative Tracking of Multicellular Tumor Spheroid Growth
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Cellular dsDNA from each cell remained constant. To trace the proliferation of MCTS, the cell number of the spheroid was determined by quantitative assessment of the amount of cellular DNA with Quant-iT PicoGreen dsDNA Reagent and Kits. The spheroids seeded at a density of 4000 cells per micro-well were collected for up to 12 days. For each sample, 1.0 ml of sodium citrate buffer solution, containing 100 mM NaCl and 50 mM sodium citrate, was added, and samples were then stored at -80°C until further analysis. The spheroids were lysed with sonication in sodium citrate solution. Then, 20 μl of the cell lysate was diluted with 80 μl Tris-EDTA buffer and incubated with 100 μl of the working solution of dsDNA reagent in 96-well plates for 2 to 5 min at room temperature. The DNA content of each sample was determined by fluorescence intensity of the mixed well with a fluorescence spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with excitation at 480 nm and emission at 520 nm. Data were analyzed by plotting fluorescence intensity versus DNA concentration.
5
Measuring Intracellular Reactive Oxygen Levels
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Reactive oxygen species (ROS) levels were measured by using a commercial kit (Beyotime, Shanghai, China). Briefly, cells were incubated with fluorescence probe DCFH-DA for at least 20 min at 37 °C. The cells were washed 3 times with serum-free cell culture medium to fully remove the extracellular DCFH-DA. Cell suspension was collected and examined for ROS by a fluorescence spectrophotometer (Thermo Scientific, Shanghai, China).
6
Measurement of Cellular ROS Generation
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The production of ROS was measured using 2, 7-dichlorodihydro fluorescent diacetate (DCFH-DA, Beyotime Institute Biotechnology). DCFH-DA passively enters the cells, cellular esterases act on the molecule to form the non-fluorescent moiety DCFH, which is ionic in nature and, therefore, trapped inside the cells. A reaction with ROS leads to an oxidation of DCFH to the highly fluorescent compound dichloroflourescein (DCF). Briefly, cells grown in 6-well plates were transfected with the negative control, GCLC siRNA-1 using Thermo Scientific Dharma FECT Transfection Reagents. One day later, cells were treated with GNPs (20μM), GNPs (20μM) and exogenous GSH (1mM) or GNPs (20μM) and ROS scavenger N-acetyl-L-cysteine (NAC, 1mM) for additional 72 h. Then the cells were washed with PBS for three times and subsequently treated with 10μM DCFH-DA. After incubated for 30minutes, the cells were trypsinized, collected by centrifugation and resuspended in PBS. The fluorescence intensity of cells (50 000) from each well was measured with a fluorescence spectrophotometer (Thermo Scientific), with excitation and emission wavelengths of 488 and 525 nm, respectively [33 (link)].
7
Quantifying Hepatocyte ROS Levels
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The rate of hepatocyte ROS generation induced by As and AsNPs was determined by adding dichlorofluorescin diacetate (DCFH-DA) to the incubated hepatocytes.19 (link) DCFH-DA penetrates hepatocytes and are hydrolyzed to form non-fluorescent dichlorofluorescin (DCF). DCF then reacts with ROS to form the highly fluorescent dichlorofluorescein that effluxes the cell. Aliquots of 1 mL of cell suspension (106 cells) were taken 1 h after incubation with As and AsNPs and centrifuged for 1 min at 1000 rpm. Cells were then resuspended in 1 mL of Krebs-Henseleit media containing 1.6 μM DCFD-DA and incubated at 37 °C for 10 min. The fluorescence intensity of dichlorofluorescein was measured using a Thermo Scientific fluorescence spectrophotometer at an excitation wavelength of 500 nm, and an emission wavelength of 520 nm.
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The rate of hepatocyte ROS generation induced by As and AsNPs was determined by adding dichlorofluorescin diacetate (DCFH-DA) to the incubated hepatocytes.19 (link) DCFH-DA penetrates hepatocytes and are hydrolyzed to form non-fluorescent dichlorofluorescin (DCF). DCF then reacts with ROS to form the highly fluorescent dichlorofluorescein that effluxes the cell. Aliquots of 1 mL of cell suspension (106 cells) were taken 1 h after incubation with As and AsNPs and centrifuged for 1 min at 1000 rpm. Cells were then resuspended in 1 mL of Krebs-Henseleit media containing 1.6 μM DCFD-DA and incubated at 37 °C for 10 min. The fluorescence intensity of dichlorofluorescein was measured using a Thermo Scientific fluorescence spectrophotometer at an excitation wavelength of 500 nm, and an emission wavelength of 520 nm.
8
GUS Activity Assay in Recombinant Apples
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Apple calli carrying the recombinant plasmids were stained with a GUS Staining Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions and GUS activity was determined with a fluorescence spectrophotometer (ThermoFisher Scientific).
9
Mitochondrial Membrane Potential Assay
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MMP was detected with JC-1 (Solarbio); 20 µl of JC-1 solution (0.15 mM in DMSO) was added to 100 μl of each suspension and was incubated at 37°C for 20 min; then, JC-1 monomer was detected at 490 nm excitation and 530 nm emission wavelengths, and JC-1 polymer was detected at 5,250 nm excitation and 590 nm emission wavelengths using a fluorescence spectrophotometer (Thermo Scientific).
10
Intestinal Permeability Assessment Using FITC-Dextran
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The intestinal permeability was assessed using an in vivo fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, MO, USA) as previously described [28 (link)]. Briefly, the mice were deprived of food and water for four hours and administrated with FITC-dextran (600 mg/kg body weight, 120 mg/mL). The plasma samples were collected after four hours, and fluorescence intensity was measured using a fluorescence spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation/emission wavelength of 485 nm/535 nm. The FITC-dextran concentrations were determined using a standard curve generated with a serial dilution of FITC-dextran nontreated plasma with PBS.
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