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Fluorescence spectrophotometer

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The Fluorescence Spectrophotometer is a laboratory instrument used to measure the fluorescence emission spectrum of a sample. It excites the sample with a specific wavelength of light and detects the intensity of the emitted light at different wavelengths.

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Lab products found in correlation

Reactive oxygen species assay kit, by Beyotime (6 mentions) Picogreen, by Thermo Fisher Scientific (2 mentions) Neuraminidase assay kit, by Beyotime (2 mentions) Mw 4000, by Merck Group (2 mentions) Dcfh da, by Merck Group (2 mentions) Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (1 mentions) Hoechst 33258 dye, by Thermo Fisher Scientific (1 mentions) Easivial ps h 4ml, by Agilent Technologies (1 mentions) Uv vis spectrophotometer, by Agilent Technologies (1 mentions) Jem 2100 lab6 transmission electron microscope, by JEOL (1 mentions) Ecosec hlc 8320 gpc, by Tosoh (1 mentions) 400 mhz spectrometer, by Bruker (1 mentions) Halotag tmr ligand, by Promega (1 mentions) Jc 1 fluorescent probe, by Beyotime (1 mentions) Multiskan go microplate reader, by Thermo Fisher Scientific (1 mentions) Zetasizer nano z instrument, by Malvern Panalytical (1 mentions) Sephadex g 25 column, by GE Healthcare (1 mentions) Novocyte flow cytometer, by Agilent Technologies (1 mentions) Novoexpress software, by Agilent Technologies (1 mentions) Mitosox, by Thermo Fisher Scientific (1 mentions)

60 protocols using fluorescence spectrophotometer

1

Quantification of Serum Biomarkers

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The serum concentrations of NE and MPO were measured using ELISA kits (eBioscience, Vienna, Austria). Briefly, the serum samples were thawed at room temperature, diluted 50-fold in the supplied sample diluent, and then added to precoated ELISA plates. Assays were then performed according to the manufacturer’s protocol. The absorbance of each microwell was read using a fluorescence spectrophotometer (Varian, Palo Alto, CA, USA) at the primary wavelength of 450 nm. The absorbance of both samples and human MPO or NE standards was determined. The concentrations of serum human MPO or NE were calculated according to the standard curve.
The serum level of double-stranded DNA (dsDNA) was quantified using the Quant-iT™ PicoGreen dsDNA reagent (P11496; Invitrogen, Carlsbad, CA, USA). Serum was diluted 10-fold in the working solution and incubated for 5 min at room temperature in the dark. The absorbance of samples was read at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using a fluorescence spectrophotometer (Varian, California, USA). The fluorescence was calculated by subtracting the fluorescence value of the reagent blank from that of each of the samples. The concentration of dsDNA was determined using the dsDNA standard curve.
Wang Y., Yang M., Xu Y., Yan S., Jin E, & Li X. (2023). Neutrophil extracellular trap burden correlates with the stenosis of coronary atherosclerosis. PeerJ, 11, e15471.
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2

Quantifying Cellular and Extracellular Matrix Components

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The biochemical compositions of the native and decellularized mECM, BMSCs in mECM and Cowhide collagen hydrogels were quantified for DNA, sulfated glycosaminoglycan (GAGs), and total collagen contents. Briefly, samples were rinsed with PBS, minced and digested with proteinase K (60 μg/mL) at 56 °C for 10 h. The content of DNA in 3D constructs was stained by Hoechst 33258 dye and absorbance was measured at 460 nm by using fluorescence spectrophotometer (Bio-Tek Instruments, USA). Calf thymus DNA was used as the standard. Intracellular GAGs secretion was analyzed using 1,9-dimethylmethylene blue assay (DMMB, Sigma, USA) and the absorption at wavelength of 525 nm was recorded on fluorescence spectrophotometer (Bio-Tek Instruments, USA), and chondroitin sulfate (Sigma, USA) was used as a standard. Finally, intracellular GAGs secretion was normalized to the DNA content of the cells and expressed as GAGs/DNA. Collagen content was quantified by measuring total hydroxyproline content using a Hydroxyproline Assay Kit (Sigma aldrich, USA). A hydroxyproline: collagen ratio of 1:7.69 was assumed to determine the collagen content [23 ].
Zhong G., Yao J., Huang X., Luo Y., Wang M., Han J., Chen F, & Yu Y. (2020). Injectable ECM hydrogel for delivery of BMSCs enabled full-thickness meniscus repair in an orthotopic rat model. Bioactive Materials, 5(4), 871-879.
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3

Intrinsic Fluorescence Assay of α-Synuclein

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Intrinsic fluorescence assay was performed with a fluorescence spectrophotometer (Varian, Cary eclipse, Australia). Aliquots of α-syn samples either alone or with vitamin K1 incubated with the single dose of SWCNT for 24 hrs were then removed and diluted ten-fold with 20 mM phosphate buffer pH 7.4 at final concentrations of 5 µM. The excitation was fixed at 270 nm (slit width: 10 nm), and intensity was recorded from 290 to 370 nm (slit width: 10 nm). The resulting spectra were corrected against buffer blank, SWCNT solution, vitamin solution, and inner filter effects.
Naskhi A., Jabbari S., Othman G.Q., Aziz F.M., Salihi A., Sharifi M., Sari S., Akhtari K., Abdulqadir S.Z., Alasady A.A., Abou-Zied O.K., Hasan A, & Falahati M. (2019). Vitamin K1 As A Potential Molecule For Reducing Single-Walled Carbon Nanotubes-Stimulated α-Synuclein Structural Changes And Cytotoxicity. International Journal of Nanomedicine, 14, 8433-8444.
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4

Quantifying ALA-Induced PpIX Fluorescence

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Beta cells were incubated with 1 mmol/l ALA for 0–24 h or 0–2 mmol/l ALA for 9 h. Then, the fluorescence of ALA-protoporphyrin IX (PpIX) was detected with a fluorescence spectrophotometer (Varian, USA) at λEx/Em = 405/635 nm. Next, PicoGreen (Thermo Fisher, USA) staining was employed to quantitate double-stranded DNA (cell population). Cells were incubated with PicoGreen at 37 °C for 1 h following incubation with ddH2O for 1 h. The fluorescence intensity of PicoGreen was measured at λEx/Em = 480/545 nm. The ALA-PpIX fluorescence was normalized to the cell population and corresponding control.
Guo T., Liu T., Sun Y., Liu X., Xiong R., Li H., Li Z., Zhang Z., Tian Z, & Tian Y. (2019). Sonodynamic therapy inhibits palmitate-induced beta cell dysfunction via PINK1/Parkin-dependent mitophagy. Cell Death & Disease, 10(6), 457.
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5

pH-Responsive PTMS Nanomicelle Preparation

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PTMS nanomicelles were prepared via self-assembly processes. Briefly, dried PTMS polymer (20mg) was dissolved in DMSO (1ml) and dialyzed against distilled water (10ml) for 24h. During this time, dialysate was frequently replaced with fresh water every three hours. After that, PTMS nanomicelles were formed and stored in 4℃. To evaluate pH-sensitive property, PTMS (20mg) and Nile Red (50μg) were dissolved in DMSO (1ml) and formed Nile Red loaded micelles after dialyzed against distilled water. Fluorescence intensities of the PTMS NPs were measured at given time-points by Varian fluorescence spectrophotometer in various buffer, pH from 7.4 to 4.0. The hydrolysis behavior of PTMS NPs was estimated by fluorescent changes.
Du L., Zhou J., Meng L., Wang X., Wang C., Huang Y., Zheng S., Deng L., Cao H., Liang Z., Dong A, & Cheng Q. (2017). The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy. Theranostics, 7(14), 3432-3445.
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6

Intracellular ROS Measurement Post-PDT

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Intracellular ROS content was determined by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF). 2′-7′-dichlorofluorescein diacetate (DCFH-DA) was added to the medium of cells at a final concentration of 20 μM and incubated at 37°C for 30 minutes 6 hours following PDT treatment or after the indicated times following PDT treatment. The cells were then washed twice with PBS carefully. Immediately after washing, the cells were measured using a fluorescence spectrophotometer (Varian Australia Pty Ltd) at 488 nm excitation and 525 nm emission wavelengths. Experiments were repeated three times independently.
Zhu X., Wang H., Zheng L., Zhong Z., Li X., Zhao J., Kou J., Jiang Y., Zheng X., Liu Z., Li H., Cao W., Tian Y., Wang Y, & Yang L. (2015). Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway. International Journal of Nanomedicine, 10, 3719-3736.
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7

Fluorescence-based Monitoring of DiY Formation

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To monitor the formation of DiY, fluorescence intensity at 400–420 nm was measured using a fluorescence spectrophotometer (Varian Ltd., Oxford, UK), using a 1-cm path length quartz cuvette (Starna Scientific, Essex, UK). Data was collected using a fluorescent excitation wavelength of 320 nm and emission collected between 340 and 600 nm, with DiY peak signal expected between 400 and 420 nm. Tyrosine fluorescence signal was monitored using an excitation wavelength of 280 nm and emission between 290–600, with the peak tyrosine emission observed at 305 nm. For experiments involving MCO, the reaction was quenched using EDTA at a final concentration of 2 mM. For all the measurements, the excitation and emission slits were both set to 10 nm with a scan rate set to 300 nm/min with 2.5 nm data intervals and an averaging time of 0.5 s. The photomultiplier tube detector voltage was 500 V.
Maina M.B., Al-Hilaly Y.K., Burra G., Rickard J.E., Harrington C.R., Wischik C.M, & Serpell L.C. (2021). Oxidative Stress Conditions Result in Trapping of PHF-Core Tau (297–391) Intermediates. Cells, 10(3), 703.
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8

Cell Attachment Quantification using Hoechst Dye

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Example 8

A previously developed cell attachment assay was used to determine the total number of cells attached to the scaffold [1]. At each time point, scaffolds were first washed in PBS to remove any unattached cells and then placed in a papain solution to digest the scaffold and lyse the cells to liberate their DNA. A Hoechst 33258 dye (Invitrogen, Carlsbad, Calif.) was used to fluorescently label double-stranded DNA [38] and fluorescence levels from each sample were read using a fluorescence spectrophotometer (Varian, Santa Clara, Calif.): 352 nm excitation, 461 nm emission. Experimental readings were then compared to a standard curve created by measuring the fluorescence levels for a range of known cell numbers to determine cell attachment at each time point as a percentage of the total number of seeded cells.

US10736992B2. Biomolecular patterning of three dimensional tissue scaffolds (2020-08-11). None [US], None [US], None [US], None [US]. Inventors: Ryan C. Bailey [US], Brendan A. Harley [US], Teresa A. Martin [US], Steven R. Caliari [US].
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9

Tryptophan Fluorescence Analysis of M-CoV-S

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A Varian Fluorescence spectrophotometer was used to measure the tryptophan fluorescence of the M-CoV-S. M-CoV-S was excited at 280 nm wavelength and emission spectra were recorded from 290 nm to 500 nm with a slit width of 5 nm. Three independent spectra were recorded and temperature was maintained by the attached Peltier temperature controller. Further, for thermal analysis the temperature was increased,20 ,21 (link) and emission spectra were recorded for each temperature.
Ahuja R., Vishwakarma P., Raj S., Kumar V., Khatri R., Lohiya B., Saxena S., Kaur G., Singh G., Asthana S., Ahmed S, & Samal S. (2024). Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen. Human Vaccines & Immunotherapeutics, 20(1), 2351664.
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10

Deubiquitinating Activity Assay of USP5

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The deubiquitinating activity of USP5 was measured for cell lysates or the components from GST-DC-UbP pull-down by using Ub-AMC substrate as described previously [18] (link), [19] (link). All the experiments were performed in a buffer of 50 mM Tris (pH 8.0) 150 mM NaCl, 10 µg/mL ovalbumin and 1 mM DTT. Ub-AMC (250 nM) was incubated with various samples for measuring the hydrolytic activities. The fluorescence of AMC release was recorded on a Fluorescence Spectrophotometer (Varian Cary Eclipse) during the reaction process with an excitation at 380 nm and the emission at 460 nm.
Song A.X., Yang H., Gao Y.G., Zhou C.J., Zhang Y.H, & Hu H.Y. (2014). A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes. PLoS ONE, 9(9), e107509.
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