Intracellular ROS production was measured using a ROS assay kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. Briefly, the cells were seeded at 24-well plates and treated by different concentrations of SANG or NAC for different periods of time. The cells were added with 10 mM DCFH-DA and incubated for 30 min at 37°C. The ROS production in the cells was determined by measuring the OD values at excitation/emission wavelengths of 488/525 nm using a fluorescence spectrophotometer (Shimadzu, Japan).
Fluorescence spectrophotometer
Manufactured by Shimadzu
Sourced in Japan
The Fluorescence Spectrophotometer is an analytical instrument designed to measure the fluorescence properties of samples. It excites molecules within a sample using a light source and then detects the emitted light, providing information about the sample's molecular composition and structure.
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Lab products found in correlation
Ros assay kit, by Beyotime (1 mentions)
U 3010 uv vis spectrophotometer, by Hitachi (1 mentions)
Uv 3600 plus spectrometer, by Shimadzu (1 mentions)
Mass spectrometer, by Thermo Fisher Scientific (1 mentions)
M205 fa, by Leica camera (1 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Infinite e plex, by Tecan (1 mentions)
Tcs sp5 2, by Leica camera (1 mentions)
Reactive oxygen species assay kit, by Nanjing Jiancheng (1 mentions)
Fd40s, by Merck Group (1 mentions)
Fourier transform infrared spectrometer, by Thermo Fisher Scientific (1 mentions)
Powder x ray diffractometer, by Malvern Panalytical (1 mentions)
Transmission electron microscope, by JEOL (1 mentions)
Uv vis spectrophotometer, by MAPADA (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Uv spectrophotometer, by Shimadzu (1 mentions)
Fluorescein isothiocyanate labeled dextran, by Merck Group (1 mentions)
96 well microplate reader, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate, by Merck Group (1 mentions)
21 protocols using fluorescence spectrophotometer
1
Intracellular ROS Measurement Protocol
2
Titration Monitoring of SSB-ssDNA Binding
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Titrations to monitor the binding of SSBf to ssDNA were performed by monitoring the fluorescence enhancement at 25°C, using a Shimadzu fluorescence spectrophotometer set at an excitation wavelength of 495 nm and an emission wavelength of 520 nm. Excitation and emission slits were set to a bandwidth of 3 and 10 nm, respectively. The concentration of SSBf was 100 nM (tetramer). Titrations were performed in 20 mM TrisOAc (pH 8.0), 1 mM DTT, and the indicated concentration of salt. The fluorescence values were corrected for dilution and normalized to the fold increase in fluorescence (fluorescence intensity of SSBf plus ssDNA divided by the SSBf fluorescence in the absence of ssDNA). The site size of SSB was determined by fitting the data to a two-segment line, where the x- and y-intercepts of the first segment and the slope of the second segment were constrained to zero. The x-intercept between the segments was taken to be the stoichiometric breakpoint of the titration. Data fitting was performed using GraphPad Prism version 5.0d. All equilibrium titrations were performed in triplicate and report the mean and standard deviation from each experiment.
3
Fluorescent Dye Binding Assay for Surface Hydrophobicity
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FCTs-JG surface hydrophobicity (H0) was assessed by measuring the interaction with the hydrophobic fluorescent dye of ANS, following a method with slight modifications by (Jiang et al., 2022c (link)). Specifically, 20 μL of an 8 mM ANS solution was added to 200 μL of 0.01, 0.05, 0.1, 0.15, and 0.2 mg/mL sample solutions, prepared in neutral PBS (10 mM). FL intensity was recorded at 390 nm (excitation) and 470 nm (emission) using a Shimadzu fluorescence spectrophotometer (Tokyo, Japan). The initial slope of fluorescence intensity versus protein concentration plot was utilized as the index of H0.
4
Spectroscopic Investigation of DNA Binding
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The absorption titration spectra were recorded on a U3010 UV-Vis spectrophotometer (Hitachi, Tokyo, Japan) by sequential addition of a specified volume of ctDNA stock solution into a 1 cm path length cuvette containing V18 solution (10 μM). After every addition of ctDNA solution, the absorption spectra were recorded from 190 to 600 nm. The intrinsic binding constant of compounds with ctDNA was determined by the equation from [27 (link),28 (link)].
All fluorescence spectra were recorded on a fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) using a 1 cm quartz cuvette in the wavelength range from 190 to 600 nm, and 522 nm was chosen as the excitation wavelength. Excitation and emission slit widths were set as 5 and 10 nm, respectively. The experimental data were plotted according to the Stern–Volmer equation in [29 (link),30 (link)]. In competition binding experiments, the concentrations of EB and ctDNA were 20 μM and 100 μM, respectively, while theV18 concentration was varied from 0 to 12 μM.
All fluorescence spectra were recorded on a fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) using a 1 cm quartz cuvette in the wavelength range from 190 to 600 nm, and 522 nm was chosen as the excitation wavelength. Excitation and emission slit widths were set as 5 and 10 nm, respectively. The experimental data were plotted according to the Stern–Volmer equation in [29 (link),30 (link)]. In competition binding experiments, the concentrations of EB and ctDNA were 20 μM and 100 μM, respectively, while the
5
UV-Fluorescence Spectroscopy of Complexes
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The ultraviolet spectra of powder samples for complexes 1 and 2 were obtained using UV-3600 plus spectrometer, within measurement range of 200 to 800 nm. And the fluorescence spectra for complexes 1 and 2 in solid states were measured on Shimadzu fluorescence spectrophotometer, recorded with excitation wavelength based on one of the strongest absorption band in the ultraviolet spectra. The quantum yield at room temperature was measured by Hamamatsu c9920-02G, Japan.
6
Comprehensive Analytical Techniques Protocol
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A 600 MHz nuclear magnetic resonance instrument (Bruker, Saarbrucken, Germany), mass spectrometer (Thermo Fisher, Waltham, MA, USA), High-resolution mass spectroscope (Waters, Milford, MA, USA), ultraviolet spectrophotometer (Prodigy, Hudson, CT, USA), fluorescence spectrophotometer (Shimadzu, Kyoto, Japan), high-performance liquid chromatograph equipped with a UV/Visible detector (1260 LC, Agilent, Santa Clara, CA, USA), confocal fluorescence microscope (TCS SP5 II, Leica, Wetzlar, German), stereo fluorescence microscope (M205FA, Leica, Wetzlar, German) fully automated fluorescence enzyme labelling instrument (Infinite EPlex, Tecan, Mannedorf, Switzerland), and flow cytometer (Attune Nxt, Invitrogen, Carlsbad, CA, USA) were used. All reagents were purchased from legitimate commercial sources.
7
Intracellular ROS Measurement Protocol
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The intracellular ROS levels were measured by the Reactive Oxygen species Assay Kit (Nanjing Jiancheng Bioengineering Institute, P.R. China) according to the manufacturer's instructions. In brief, the cells were collected by centrifugation at 13 700×g for 10 min, and re-suspended with PBS solution and diluted to OD 600 = 1.0. The volume of 1 mL cell suspension was added with 10 mM DCFH-DA, and incubated at 37°C for 30 min. The cells were then re-suspended in 1 mL PBS solution, and the relative fluorescence was measured at excitation and emission wavelengths of 485 and 525 nm by using a fluorescence spectrophotometer (Shimadzu, Japan). Untreated cells were used as reference, and the relative ROS amounts were showed by fluorescence intensity (9) .
8
Fluorescence-based NPN Assay
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It was determined according to the method previously described [18 (link),33 (link)]. In brief, a stock solution of 5 mM NPN in ethanol was diluted using potassium phosphate buffer (PBS) (pH 7.5) to reach a concentration of 20 μM. The fluorescence of the samples was measured using a fluorescence spectrophotometer (SHIMADZU, Kyoto, Japan) at an excitation and emission wavelength of 340 and 420 nm, respectively.
9
Fluorescence Analysis of Microparticle Interactions
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To study the interaction between components of the microparticles, the samples were analyzed using a fluorescence spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Emission fluorescence was monitored at 300−450 nm, whereas excitation fluorescence was monitored at 280 nm, with a slit width of 10 nm.
10
Characterizing AIE and AIE-cRGD Optical Properties
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The UV absorption of AIE, AIE–cRGD was measured by double beam ultraviolet spectrophotometer (Persee, Beijing, China) from 200 to 450 nm. The AIE, AIE–cRGD was diluted in various concentrations in DMSO/water (v/v = 1/100). The fluorescence intensity of AIE–cRGD was measured by fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) under an excitation of 360 nm. The AIE–cRGD was dissolved in 1×PBS to obtain a series of concentrations.
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