TMRE–Mitochondrial Membrane Potential Assay Kit (Abcam) was used to determine the MMP in C28/I2 cells, and 3 × 105 C28/I2 cells were incubated with the MMP-sensitive fluorescent TMRE for 30 min at 37°C (1000 nM FCCP was added to the positive control cells 10 min prior to TMRE). Cells were then trypsinized, centrifuged and resuspended in 0.4 ml of DPBS with 0.2% BSA and were analysed for TMRE fluorescence by the fluorescence spectrophotometer (Hitachi) with an excitation wavelength of 549 nm and an emission wavelength of 575 nm. Complex IV activity was measured by Complex IV Rodent Enzyme Activity Microplate Assay Kit (Abcam) according to manufacturer’s instructions. In brief, C28/I2 cells were collected and lysed with the detergent extraction. Then protein samples were serially diluted and were incubated at room temperature for 3 h in the plate in each well of which the enzyme-linked monoclonal antibody against complex IV has been immobilized. Then the binding was assayed by the fluorescence spectrophotometer (Hitachi) with an excitation wavelength of 549 nm and an emission wavelength of 575 nm. Results were presented as a percent level to control.
Fluorescence spectrophotometer
Manufactured by Hitachi
Sourced in Japan
The Fluorescence Spectrophotometer is a laboratory instrument used to measure the intensity of fluorescent light emitted by a sample when exposed to light of a specific wavelength. It determines the concentration of fluorescent compounds in a sample by analyzing the intensity of the emitted fluorescent light.
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Lab products found in correlation
Tmre mitochondrial membrane potential assay kit, by Abcam (2 mentions)
Complex 4 rodent enzyme activity microplate assay kit, by Abcam (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Lucifer yellow, by Merck Group (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Na assay kit, by Beyotime (1 mentions)
H 7650 transmission electron microscope, by Hitachi (1 mentions)
Fv10 asw, by Olympus (1 mentions)
Axis ultra dld, by Shimadzu (1 mentions)
Uv 3600 uv vis nir spectrophotometer, by Shimadzu (1 mentions)
S 4800, by Hitachi (1 mentions)
Jem 2100f, by JEOL (1 mentions)
Multimode 8, by Bruker (1 mentions)
D8 x ray diffractometer, by Bruker (1 mentions)
Tristar 3020 analyzer, by Micromeritics (1 mentions)
Fls920, by Edinburgh Instruments (1 mentions)
Biochemical assay kit, by Nanjing Jiancheng (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (1 mentions)
Dcfh da kit, by Solarbio (1 mentions)
51 protocols using fluorescence spectrophotometer
1
Mitochondrial Membrane Potential and Complex IV Activity in C28/I2 Cells
2
Mitochondrial Function Evaluation in C28/I2 Cells
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A TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK) was used to determine the mitochondrial membrane potential (MMP) in the C28/I2 cells. C28/ I2 cells (3 x 10 5 ) were incubated with the MMP-sensitive fluorescent TMRE for 30 minutes at 37°C (1000 nM FCCP was added to the positive control cells 10 min prior of TMRE). Cells were then trypsinized, centrifuged, and resuspended in 0.4 mL Dulbecco's phosphatebuffered saline with 0.2% bovine serum albumin and analyzed for TMRE fluorescence using the fluorescence spectrophotometer (Hitachi, Tokyo, Japan) with an excitation wavelength of 575 nm and an emission wavelength of 549 nm. Complex IV activity was measured using a Complex IV Rodent Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, UK) according to the manufacturer instructions. In brief, C28/I2 cells were collected and lysed with the extraction detergent. The protein samples were then serially diluted and incubated at room temperature for 3 hon the plate, in each well of which the enzyme-linked monoclonal antibody against complex IV has immobilized. The binding was then assayed using the fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 575 nm and an emission wavelength of 549 nm. The results are reported as a percent level of the control.
3
Cellulase Activity and Fluorescence Assay
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Samples were centrifuged at 8000 × g for 10 min to collect supernatants for cellulase activity and fluorescence intensity measurement. Cellulase activity assays were carried out as previously described (19 (link), 21 (link), 23 (link), 49 (link)). Fluorescence intensity was determined at excitation/emission wavelengths of 540/635 nm under a fluorescence spectrophotometer (Hitachi Ltd., Japan). The glucose concentration was measured using a glucose detection kit (Shanghai Rongsheng Biotech, China).
4
Neuraminidase Activity Assay for NDV
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NA activity was tested using the NA assay kit (Beyotime, China). According to the manufacture instructions, 50 ng of HN protein or 10 μl of allantoic fluids infected with NDV was mixed with 70 μl of detection buffer, 10 μl of fluorogenic substrate and 10 μl of water. Then the mixture was incubated at 37°C for 30 min, followed by detecting the NA fluorogenic substrate cleaved by virus or protein, using a Fluorescence Spectrophotometer (Hitachi, F-7000) with an excitation wavelength of 322 nm and an emission wavelength of 450 nm. NA activity was represented as fluorescence intensity of samples above that of corresponding solutions without virus or protein.
5
Characterizing mCherry Fluorescence with LaMs
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The emission spectra of mCherry with LaMs were measured while the excitation wavelength was fixed at 560 nm. Additionally, the excitation spectra were measured while the emission wavelength was set at 610 nm. The pure mCherry protein without LaM was used as the control group. The final concentration of mCherry in all groups was equal, and SUMO-LaMs were added as the molar ratio of LaM to mCherry as 1.25:1, and then the sample was diluted with GF buffer. The spectra were obtained with a fluorescence spectrophotometer (Hitachi, Japan). The emission spectra were acquired between 580 nm and 700 nm. The spectra were analyzed with OriginPro software 2021.
6
Fluorescence Spectroscopy of Caseinate-Polyphenol
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Three-dimensional fluorescence spectra of the caseinate (15 µmol/L) and caseinate-polyphenol complex (15 µmol/L for both) were monitored at 293 K using the fluorescence spectrophotometer (Hitachi, Kyoto, Japan) and 10 mm quartz cuvette. The emission and excitation wavelengths were recorded from 210–400 and 210–350 nm, respectively. The slits of emission and excitation were both fixed at 5 nm while the scanning rate was set at 240 nm/min. Other parameters were the same as those used in the assay of fluorescence spectra.
7
Mitochondrial Superoxide and ROS Quantification
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The mitochondrial superoxide was examined with MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen) (Ex/Em =580/510 nm), which was highly selective and detects the superoxide in the mitochondria. C28/I2 cells were incubated with 5 μM MitoSOX™ Red at 37°C for 30 min and then were detected after the removal of the reagent and three times washing. Intracellular ROS level of was quantified by the ROS-sensitive fluorophore 5-(and-6)-chloromethyl-2, 7-dichlorodi-hydrofluorescein diacetate (Invitrogen) according to the product’s manual. Briefly, confluent C28/I2 cells were incubated with the 5-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate probe at 37°C for 30 min and washed with PBS for three times. Then the cells were rinsed with HBSS and were measured using the fluorescence spectrophotometer (Hitachi) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Results were presented as a percent level to control.
8
Seedling Greening and Chloroplast Analysis
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Seedling greening was analyzed by exposing 5-day-old dark-grown seedlings to white light (16 h-light/8 h-dark) for 2 days. Chlorophyll autofluorescence was analyzed using a confocal laser scanning microscope (Olympus, FV10-ASW). In the PAC-treated etiolated seedlings, 0.01 µM of PAC was used. Greening ratio was determined by counting the percentage of green cotyledons of each genotype. Pchlide was extracted from 5-day-old etiolated seedlings with 1 mL of ice-cold 80% acetone in the dark. The samples were centrifuged at 13000 rpm for 10 min, and fluorescence was excited by the wavelength of 440 nm and scanned from 600 nm to 700 nm using a fluorescence spectrophotometer (Hitachi) at room temperature [54] (link). The results were presented by relative fluorescence per seedling. Chlorophyll was measured as described previously [55] (link). The Fv/Fm parameter was measured using light-stressed leaf discs after 15-min adaptation to darkness [56] (link). For electron microscopy observation, cotyledons of 5-day-old etiolated and 25-day-old seedlings were fixed and processed as previously described [57] , and examined with an H-7650 transmission electron microscope (Hitachi).
9
Comprehensive Material Characterization Protocol
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Powder X-ray diffraction (XRD) measurements were acquired using a Bruker D8 X-ray diffractometer (Bruker, Germany) with Ni-filtered Cu Kα radiation (40 kV, 40 mA). Atomic force microscopy (AFM) images were obtained by using a Multimode 8 (Bruker, Germany) in the tapping mode. The size and morphology of the products were characterized by a field-emission scanning electron microscope (FESEM, S-4800, HITACHI, Japan) and transmission electron microscope (TEM, JEM-2100F, JEOL, Japan). Fourier transform infrared spectroscopy (FTIR) was determined by a Thermo Fisher Nicolet 6700 spectrometer (ThermoFisher, USA). X-ray photoelectron spectroscopy (XPS) measurements were recorded on KRATOS Axis Ultra Dld (SHIMADZU, Japan) equipped with a monochromatic X-ray source (Al Kα, hν = 1486.6 eV). UV-vis diffused reflectance spectra (UV-vis DRS) of the samples were analyzed using a UV-3600 UV-vis-NIR spectrophotometer (SHIMADZU, Japan) at room temperature. Photoluminescence (PL) spectrum was recorded on a on a Fluorescence Spectrophotometer (F-7000, HITACHI, Japan) at an excitation wavelength of 380 nm. The transient state fluorescence spectra were tested by fluorescence spectrometer (FLS920, Edinburgh Instruments, UK). Nitrogen adsorption isotherm measurements were carried out at 77 K with a Micromeritics Tristar 3020 analyzer (USA).
10
Quantifying Glutathione and MDA in Plant Leaves
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The amounts of GSH in plant leaves were measured according to Hissin and Hilf [44 (link)], with modifications according to Jiang et al. [1 (link)]. Frozen leaf tissues were extracted with 100 mmol·L−1 sodium phosphate-EDTA buffer (pH 8.0) and 6.5% trichloroacetic acid. The homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C. GSH levels in the supernatant were measured fluorometrically after incubation with o-phthaldialdehyde in phosphate-EDTA buffer. The fluorescence intensity was recorded at 420 nm after excitation at 350 nm on a fluorescence spectrophotometer (Hitachi, Ichige, Hitachinaka, Japan).
The amounts of MDA in plant leaves were determined using a biochemical assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.
The amounts of MDA in plant leaves were determined using a biochemical assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.
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