This was measured as previously described [21 (link)], with some modifications using a fluorescent indicator (DCFH-DA, Sigma). The purified mitochondria (0.1 mg/mL protein) were added to ice-cold potassium phosphate buffer (50 mm, pH 7.4) containing 5 mm DCFH-DA. The total volume of the suspended mitochondrial particles was maintained at 3.0 mL. DCFH-DA was incorporated into the membrane by pre-incubating at 37°C for 15 min. To remove the unloaded DCFH-DA, the mixture was centrifuged at 12 500 g and 4°C for 8 min. Subsequently, the fluorescence intensity (Ex = 488 nm, Em = 525 nm) was measured with a fluorescence spectrophotometer (Shimadzu RF5301, Kyoto, Japan).
Rf 5301
Manufactured by Shimadzu
Sourced in Japan
The RF-5301 is a fluorescence spectrophotometer designed for laboratory use. It is capable of measuring the fluorescence intensity of samples.
Automatically generated - may contain errors
Lab products found in correlation
Uv 1800, by Shimadzu (4 mentions)
Jem 2100, by JEOL (2 mentions)
D8 advance, by Bruker (2 mentions)
Thioglycolate, by Merck Group (2 mentions)
10 kda cellulose membrane, by Merck Group (2 mentions)
Nano scope 3d, by Veeco (2 mentions)
Amplex ultrared reagent, by Thermo Fisher Scientific (2 mentions)
Jc 1 assay kit, by Beyotime (2 mentions)
Uv 2600, by Shimadzu (2 mentions)
Spectrofluorometer, by Shimadzu (2 mentions)
Nano s, by Malvern Panalytical (2 mentions)
Uv 1800 spectrophotometer, by Shimadzu (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Dcfh da, by Merck Group (1 mentions)
Spectrofluorimeter, by Shimadzu (1 mentions)
J 810, by Jasco (1 mentions)
Triple quad ms, by Agilent Technologies (1 mentions)
Uv 3600, by Shimadzu (1 mentions)
Smartlab, by Rigaku (1 mentions)
Chi 660b, by Chenhua (1 mentions)
72 protocols using rf 5301
1
Mitochondrial Oxidative Stress Measurement
2
Dialysis Monitoring with Fluorescence
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39 dialysis sessions of 10 patients (age 59 ± 15 years) were followed. 100 mg B6 was routinely injected to patients after each dialysis session. The dialysis machine used was Fresenius 5008H (Fresenius Medical Care, Germany), dialyzers were FX8 or FX1000, the dialysate and blood flow varied from 500–800 mL/min and 300–350 mL/min, respectively. The dialysate samples were collected 7–10, 60, 120, 180, 240 minutes after the start of the dialysis session from the outlet dialysate line and from tank (145 HD and 50 HDF samples in total). All dialysate samples were acidified down to pH 4.25 with formic acid before the HPLC analysis for the best chromatographic separation and stable retention times. Full fluorescence spectra of spent dialysates in the range of excitation/emission 220–500 nm and emission with excitation increment 10 nm were recorded with the spectrofluorophotometer RF-5301 by Shimadzu (Kyoto, Japan). The cell with optical path 4 mm was used for measurement and the Panorama Fluorescence 1.2 software by Shimadzu for spectral data processing.
3
Measuring Mitochondrial Membrane Potential
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Mitochondrial membrane potential was estimated by fluorescence changes of safranin [37 ] recorded by a RF-5301 Shimadzu spectrofluorimeter (Kyoto, Japan) operating at excitation and emission wavelengths of 495 and 586 nm, respectively, with slit widths of 3 nm. Values of mitochondrial membrane potential (Δψm) were expressed relative to the control.
4
H+-ATPase Fluorescence Titration Protocol
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The H+-ATPase fluorescence data was obtained at 25 °C in a spectrofluorophotometer Shimadzu RF5301 equipped with a thermoregulated cell. The plasma membrane H+-ATPase (3.22 μg prot.) was added to the incubation mixture of 20 mM phosphate buffer, pH 6.8, plus 5 mM MgCl2. The ATPase intrinsic fluorescence spectra were recorded between wavelengths (λ) 300 to 450 nm using an excitation λ of 295 nm and 5 nm slit. The ATPase intrinsic fluorescence was titrated with ADP and AMP-PCP. The titration was performed by a stepwise increase of nucleotide concentration (2 μL additions from 50 mM stock solution). After each increment, the sample was incubated for 1 min, and the fluorescence spectrum was obtained. Although volume additions and nucleotide concentration did not significantly affect the final sample volume and protein excitation, the fluorescence spectra were corrected for dilution and inner filter effect [49 ] using the calculated ATP molar extinction coefficient of 16.76 M−1·cm−1 at λ of 295 nm, respectively.
5
Structural Stability Analysis of Bevacizumab
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Lyophilized eBev-DPPNs or cBev-DPNs were resuspended in PBS (20 mM, pH 7.4) and incubated at 37 °C, then bevacizumab that had dissociated from the surface of the nanoparticles was recycled and filtered with a 0.22-μm aperture for further testing.
The primary, secondary, and tertiary structural stability of bevacizumab was analyzed using size exclusion chromatography (SEC-HPLC), circular dichroism (CD) (J-810, Jasco, USA), and fluorescence spectroscopy (RF-5301, Shimadzu, Japan) as previously described.25 (link)
The primary, secondary, and tertiary structural stability of bevacizumab was analyzed using size exclusion chromatography (SEC-HPLC), circular dichroism (CD) (J-810, Jasco, USA), and fluorescence spectroscopy (RF-5301, Shimadzu, Japan) as previously described.25 (link)
6
Fluorescence Spectra Measurement Protocol
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The fluorescence spectra of samples were measured on a spectrofluorometer (Shimadzu RF-5301) at 365 nm excitation wavelength and 380-650 emission wavelength. All experiments were made using pure solvent as a reference.
7
Extracellular H2O2 Quantification via Amplex Red Assay
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Amplex red catalase assay kit was used for the extracellular H2O2 quantification in the cell culture media as per the manufacturer‘s protocol. Briefly, cells plated in 6-well plates at a density of ∼5 ×105 cells per well were treated with the appropriate concentration of EGF, HG, or EGF+HG. 50 µl of Amplex Red reagent/horseradish peroxide (HRP) working solution (50 µl of 10 mM Amplex Red reagent, 100 µl of 10 U/ml HRP stock solution, and 4.85 ml of 1X reaction buffer) was added to each well containing treated and control samples and incubated in dark for 30 min in a CO2 incubator. The fluorescence signal was determined using a spectrofluorophotometer (RF-5301; Shimadzu, Kyoto, Japan) with an excitation wavelength of 568 nm and an emission wavelength of 581 nm (Ali et al., 2021 (link); Bhat et al., 2020 (link)).
8
Fluorescence Quenching Assay for NPs
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Fluorescence
spectra were
acquired on a spectrofluorophotometer (Shimadzu, RF-5301). All solutions
were prepared in 10% (v/v) PBS without calcium and magnesium. Samples
were excited at 280 nm, and emission was collected between 250 and
500 nm using 5 nm slit widths for both excitation and emission. BSA
(9.8 μg mL–1) was measured in the presence
of nonfluorescent 60 nm carboxylate-modified and 58 nm amine-modified
NPs. Working NP concentration ranged from 67 to 533 pM. Experiments
were repeated in triplicate, and the mean and standard deviation are
plotted for each data point. Corrected spectra are the raw spectra
with the NP scatter peak and buffer contribution subtracted out and
the initial emission value at 250 nm set to zero. The Stern–Volmer
equation was used to calculate the equilibrium constant (eq3 ).
The fluorescence
intensity ratio of
BSA at λmax in the absence (Fo) and presence (F) of a
quencher is calculated and plotted versus the NP quencher concentration
([NP]). The slope of the line is equal to the Stern–Volmer
equilibrium constant (KSV, M–1). For static quenching, KSV is equal
to the equilibrium association constant.92 The first four points were fit linearly to calculate an effective
equilibrium constant.
spectra were
acquired on a spectrofluorophotometer (Shimadzu, RF-5301). All solutions
were prepared in 10% (v/v) PBS without calcium and magnesium. Samples
were excited at 280 nm, and emission was collected between 250 and
500 nm using 5 nm slit widths for both excitation and emission. BSA
(9.8 μg mL–1) was measured in the presence
of nonfluorescent 60 nm carboxylate-modified and 58 nm amine-modified
NPs. Working NP concentration ranged from 67 to 533 pM. Experiments
were repeated in triplicate, and the mean and standard deviation are
plotted for each data point. Corrected spectra are the raw spectra
with the NP scatter peak and buffer contribution subtracted out and
the initial emission value at 250 nm set to zero. The Stern–Volmer
equation was used to calculate the equilibrium constant (eq
The fluorescence
intensity ratio of
BSA at λmax in the absence (Fo) and presence (F) of a
quencher is calculated and plotted versus the NP quencher concentration
([NP]). The slope of the line is equal to the Stern–Volmer
equilibrium constant (KSV, M–1). For static quenching, KSV is equal
to the equilibrium association constant.92 The first four points were fit linearly to calculate an effective
equilibrium constant.
9
Comprehensive Characterization of Material Properties
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X-ray diffraction (XRD) data were obtained on an X-ray diffractometer
(SmartLab, Rigaku) with Cu Kα radiation in the range of 10–70°
(2θ). The morphology and microstructure of the samples were
studied by transmission electron microscopy (TEM, JEM-2100, JEOL,
Japan) and high-resolution TEM (HRTEM). The elements of the sample
prepared were analyzed by energy-dispersive spectroscopy (EDS). UV–vis
diffuse reflectance spectra (DRS) of the samples were measured using
a UV–vis spectrophotometer (UV-3600, Shimadzu). Photoluminescence
(PL) with an excitation wavelength of 325 nm was obtained using a
fluorescence spectrophotometer (Shimadzu RF-5301). The specific Brunauer–Emmett–Teller
(BET) surface areas were determined by nitrogen adsorption using Micromeritics
ASAP 2020 nitrogen adsorption apparatus. The analysis of intermediates
was performed using an HPLC-MS system (Agilent 1290/6460, Triple Quad
MS) equipped with a Zorbax XDB-C18 column (150 × 2.1 mm, 3.5
μm). The electrochemical measurement was performed with an electrochemical
workstation (CHI660B, Chen Hua Instruments, Shanghai, China).
(SmartLab, Rigaku) with Cu Kα radiation in the range of 10–70°
(2θ). The morphology and microstructure of the samples were
studied by transmission electron microscopy (TEM, JEM-2100, JEOL,
Japan) and high-resolution TEM (HRTEM). The elements of the sample
prepared were analyzed by energy-dispersive spectroscopy (EDS). UV–vis
diffuse reflectance spectra (DRS) of the samples were measured using
a UV–vis spectrophotometer (UV-3600, Shimadzu). Photoluminescence
(PL) with an excitation wavelength of 325 nm was obtained using a
fluorescence spectrophotometer (Shimadzu RF-5301). The specific Brunauer–Emmett–Teller
(BET) surface areas were determined by nitrogen adsorption using Micromeritics
ASAP 2020 nitrogen adsorption apparatus. The analysis of intermediates
was performed using an HPLC-MS system (Agilent 1290/6460, Triple Quad
MS) equipped with a Zorbax XDB-C18 column (150 × 2.1 mm, 3.5
μm). The electrochemical measurement was performed with an electrochemical
workstation (CHI660B, Chen Hua Instruments, Shanghai, China).
10
Mitochondrial ROS Measurement with DCFH-DA
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RS levels were measured using the oxidant sensing fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) [36 (link)]. The oxidation of DCFH (which is formed by the action of esterase on DCFH-DA) to fluorescent dichlorofluorescein (DCF) was determined at 488 nm for excitation and 525 nm for emission. An aliquot of 5 μL (50 μg protein) of the homogenate of the isolated mitochondria was added to 3 mL of buffer III (containing 5 mM succinate). The reaction medium was exposed to PBE (25, 50, or 100 μg/mL) or catechin (1, 5, or 10 μg/mL) and/or 80 μM Ca2+/150 μM SNP. After 10 s, 10 μM (DCFH-DA) (prepared in ethanol) was added to the mixture and the fluorescence intensity from DCF was measured for 300 s using a spectrofluorimeter (RF-5301 Shimadzu, Kyoto, Japan).
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