Luminescence spectrometer ls 50

Manufactured by PerkinElmer

Sourced in United States

The Luminescence Spectrometer LS 50 is a laboratory instrument used for the measurement and analysis of luminescence, including fluorescence and phosphorescence. It provides precise and sensitive detection of light emission from samples.

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Lab products found in correlation

Fl winlab software package, by PerkinElmer (1 mentions) Nigericin, by Merck Group (1 mentions) Monensin, by Merck Group (1 mentions) Lysosensor yellow blue dextran, by Thermo Fisher Scientific (1 mentions) Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)

11 protocols using luminescence spectrometer ls 50

Determination of Kidney Tissue Thiols

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Free sulfhydryl groups were determined using Ellman’s method (Ellman 1970 ). Ellman’s reagent (DTNB; 5,5′-dithiobis-(2-nitrobenzoic) acid) served as a chromogen to measure the thiol levels.
Fifty mg portions of kidney tissue were cut into small pieces and homogenized in 2 mL of ice-cold 0.1 M PBS. The homogenates were centrifuged for 10 min at 5000 ×g at 4 °C. Then, the Ellman’s reagent was added to the above supernatant.
The absorbance of the obtained solution was measured at 412 nm using a LS-50 Luminescence Spectrometer (Perkin Elmer, Norwalk, USA). Concentrations of −SH groups were expressed as μM.

Skibska B., Kochan E., Stanczak A., Lipert A, & Skibska A. (2023). Antioxidant and Anti-inflammatory Effects of α-Lipoic Acid on Lipopolysaccharide-induced Oxidative Stress in Rat Kidney. Archivum Immunologiae et Therapiae Experimentalis, 71(1), 16.

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Kinetic and Inhibitor Coefficients of MMP Peptide Substrates

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To determine the kinetic and inhibitor kinetic coefficients K_m, and K_i, initial rate experiments were performed using a Perkin Elmer LS 50 Luminescence spectrometer and the FL WinLab Software Package (Perkin Elmer). The reactions were followed for one minute and during that time 600 data points were collected. The excitation and emission wavelengths for the two fluorescence quenched MMP peptide substrates, McaPLGLDpaAR-NH₂ and Mca-RPPGFSAFK(Dpn)-OH were; λ_ex = 320 nm, λ_em = 405 nm and a slit width = 10 nm at both wavelengths. All assays were performed at 37°C in an assay buffer of 0.1 M Hepes pH 7.5, 0.005% Brij-35, 10 mM CaCl₂ and a total assay volume of 100 μl.

Sylte I., Dawadi R., Malla N., von Hofsten S., Nguyen T.M., Solli A.I., Berg E., Adekoya O.A., Svineng G, & Winberg J.O. (2018). The selectivity of galardin and an azasugar-based hydroxamate compound for human matrix metalloproteases and bacterial metalloproteases. PLoS ONE, 13(8), e0200237.

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Intrinsic Fluorescence Titration of FXR-LBD Interactions

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A LS 50 luminescence spectrometer (Perkin Elmer) was used to measure steady state fluorescence. The monochromator slit widths for excitation and emission were both set to 3.5 nm. For the fluorescence titrations, after each addition of ligand, the protein sample was excited at 295 nm to minimize the fluorescence contribution of tyrosine residues and the emission spectra were acquired from 305 nm to 480 nm. The ligand-FXR-LBD interactions were studied by using an intrinsic fluorescence titration method in which 400 μL of 10 μM protein (dissolved in 50 mM sodium phosphate buffer containing 0.5 M NaCl, 0.5 mM CHAPS, 1 mM TCEP, 0.5 M sucrose and 10% glycerol, pH 7.4) was titrated with 1000 μM ligand (dissolved in DMSO) until the ligand/protein ratio reached 5 to 1. Before analysis of the binding and quenching data, fluorescence spectra were corrected (F_cor) for the background fluorescence of the specific ligand F_B, the instrument-dependent monochromator and photomultiplier response emission correction factor F_S, and the dilution factor A: F_cor = (F₀ − F_B) × 100×A/F_S where F₀ is the measured fluorescence intensity. All measurements were performed at 25°C and repeated three times. Fluorescence quenching data analysis procedure was described in Text S1.

Yang L., Broderick D., Campbell Y., Gombart A.F., Stevens J.F., Jiang Y., Hsu V.L., Bisson W.H, & Maier C.S. (2016). Conformational Modulation of the Farnesoid X Receptor by Prenylflavonoids: Insights from Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), Fluorescence Titration and Molecular Docking Studies. Biochimica et biophysica acta, 1864(12), 1667-1677.

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Purification and Characterization of Recombinant Bovine OBP

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The recombinant form of bovine OBP tagged with six histidine residues at the amino terminal (6-His-bOBP) was obtained from a BL21-DE3 E. coli strain transformed with the expression vector pT7-7 containing the appropriate OBP cDNA, as previously reported.^{41 (link)} Purification of the protein was carried out by affinity chromatography using Ni-NTA agarose according to the instructions of the manufacturer, followed by dialysis in 50 mM sodium phosphate pH = 7.3. Protein purity was assessed by SDS-PAGE and its concentration was determined by measuring the absorbance value at 280 nm.
Functionality of the protein in solution was determined by direct titrations using the fluorescent ligand 1-aminoanthracene (AMA), as previously reported.^{18 (link)} Briefly, samples of 0.5 μM 6-His-bOBP in 20 mM Tris–HCl buffer pH = 7.3 were incubated overnight at 4 °C with increasing concentrations of AMA (0.15–20 μM). Fluorescence emission spectra were recorded between 450 and 550 nm with a PerkinElmer LS 50 luminescence spectrometer (PerkinElmer, Waltham, MA, USA) (excitation and emission slits at 5 nm) at a fixed excitation wavelength of 380 nm. The formation of the binding complex between 6-His-bOBP and AMA was monitored as an increase in the fluorescence emission intensity at 480 nm.

Riboni N., Spadini C., Cabassi C.S., Bianchi F., Grolli S., Conti V., Ramoni R., Casoli F., Nasi L., de Julián Fernández C., Luches P, & Careri M. (2021). OBP-functionalized/hybrid superparamagnetic nanoparticles for Candida albicans treatment. RSC Advances, 11(19), 11256-11265.

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Proteasome Activity Assay in COLIV and COL1 Cells

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Cells were plated over COLIV or COLI at indicated times. Then, they were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 2 mM DTT and 2 mM ATP). Samples were frozen at −80°C and thawed three times and then centrifuged for 10 min. at 10,000 × g. Proteasome activity was measured in the supernatant fraction using the fluorogenic substrate (80 μM) Succ‐LLVY‐AMC. Equal fractions of the supernatants were pre‐incubated with the proteasome inhibitor MG132 (10 μM), for 30 min. at 37°C before addition of the substrate. The fluorescence was determined by Luminescence spectrometer LS50 (Perkin Elmer, Wellesley, MA, USA) (380 nm excitation wavelength and 460 nm emission wavelength). Activities of enzymes were measured as arbitrary unit/min/mg protein and then expressed as percentage of the activity found in cells plated on COLIV, used as control cells.

Olmos G., Muñoz‐Félix J.M., Mora I., Müller A.G., Ruiz‐Torres M.P., López‐Novoa J.M, & Rodríguez‐Puyol D. (2017). Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition. Journal of Cellular and Molecular Medicine, 22(1), 302-314.

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Quantifying Endo-lysosomal pH Using Ratiometric Dye

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Endo-lysosomal pH was measured using a ratiometric pH indicator dye, LysoSensor™ Yellow/Blue dextran (Molecular Probes/Life Technologies), a dual excitation dye that allows pH measurement in endocytic organelles independently of dye concentration. Lymphoblasts (4 × 10⁶) were incubated for 3 h at 37°C with 0.5 mg/ml of LysoSensor™ Yellow/Blue dextran in culture medium. Cells were divided into five samples, four to perform the calibration curve and one for the measurements. To obtain a pH calibration curve, cells were resuspended with MES calibration buffer solution (5 mM NaCl, 115 mM KCl, 1.2 mM MgSO₄ and 25 mM MES, pH ranging from 3.7 to 7.6) containing 10 µM monensin (Sigma-Aldrich) and 10 μM nigericin (Sigma-Aldrich). For the pH measures, cells were resuspended in MES calibration buffer solution pH 7.7 in the absence of ionophores. Emission scans were collected at 450 nm and 528 nm with the Luminescence Spectrometer LS 50 (Perkin Elmer), using excitation at 360 nm and emission/excitation bandwidths set to 4 nm. Calibration data (ratio 450/528) were fitted to a linear regression with the software GraphPad Prism5 and the sample ratios converted into absolute pH values by interpolation in the calibration function.

Fassio A., Esposito A., Kato M., Saitsu H., Mei D., Marini C., Conti V., Nakashima M., Okamoto N., Olmez Turker A., Albuz B., Semerci Gündüz C.N., Yanagihara K., Belmonte E., Maragliano L., Ramsey K., Balak C., Siniard A., Narayanan V., Ohba C., Shiina M., Ogata K., Matsumoto N., Benfenati F, & Guerrini R. (2018). De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy. Brain, 141(6), 1703-1718.

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Quantifying Hydrogen Peroxide in Tissue

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Hydrogen peroxide (H₂O₂) generation in homogenates was measured using the HRP/HVA systems. First, 50 mg of frozen tissue fragments were homogenized with 2 mL of 1.15% KCl. Then, 10 μL of homogenate were placed in 2 Eppendorf tubes. In one tube, the reaction mixture for the calibration curve of H₂O₂ was added, which contained phosphate-buffered saline (PBS) (pH 7.0) and horseradish peroxidase (HRP) (1 U/mL) containing 400 μmol homovanillic acid (HVA). In the second tube, we placed PBS and 1 U/mL HRP to determine H₂O₂.
Both tubes were simultaneously incubated for 60 min at 37 °C. Subsequently, PBS and 0.1 M glycine-NaOH buffer (pH 12.0) with 25 mM EDTA were added to each Eppendorf tube to stop the enzymatic reaction. Excitation was set at 312 nm and emission was measured at 420 nm (Perkin Elmer Luminescence Spectrometer, LS-50, Norwalk, CT, USA). Readings were converted to the H₂O₂ concentration using the regression equation prepared from 3 series of calibration experiments with 10 increasing H₂O₂ concentrations (range 10–1000 μM).

Skibska B., Goraca A., Skibska A, & Stanczak A. (2022). Effect of Alpha-Lipoic Acid on Rat Ventricles and Atria under LPS-Induced Oxidative Stress. Antioxidants, 11(4), 734.

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Quantifying Total Sulfhydryl Groups

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Total sulfhydryl groups measurement was performed using Ellman’s method [32 ]. As a chromogen, to measure thiol levels, Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoic) acid, DTNB) was used. The absorbance of the obtained solution was measured at 412 nm using the Perkin Elmer Luminescence Spectrometer LS-50 (Norwalk, CT, USA). The total concentration of thiol groups was calculated from the regression equation prepared from 3 repeats of increasing concentrations of glutathione (2–200 µM). Concentrations of -SH groups were expressed as μM.

Skibska B., Goraca A., Skibska A, & Stanczak A. (2022). Effect of Alpha-Lipoic Acid on Rat Ventricles and Atria under LPS-Induced Oxidative Stress. Antioxidants, 11(4), 734.

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H2O2 Transport in S. cerevisiae

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The YSH1172 aqy-null S. cerevisiae strain transformed with pVV214-VvXIP1 and pVV214-empty vector (control) constructs were used to study the transport of H₂O₂. Liquid cultures were grown overnight in the dark in presence of 1 μM 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA, Molecular Probes), a fluorophore sensitive to reactive oxygen species (ROS) [18 (link)]. In its acetylated form, the dye can freely diffuse into yeast cells but, once inside, the fluorochrome is deacetylated and unable to cross the membrane making it susceptible to oxidation by ROS. After incubation, cells were washed three times with MOPS buffer (pH 7.0) and resuspended in this buffer to a final OD_{600 nm} = 1.4. After the addition of 50 μM H₂O₂, the fluorescence of 2 mL yeast suspension was followed over time at 20°C in a spectrofluorometer at an excitation/emission of 492/527 nm (Perkin Elmer Luminescence Spectrometer LS 50).

Noronha H., Araújo D., Conde C., Martins A.P., Soveral G., Chaumont F., Delrot S, & Gerós H. (2016). The Grapevine Uncharacterized Intrinsic Protein 1 (VvXIP1) Is Regulated by Drought Stress and Transports Glycerol, Hydrogen Peroxide, Heavy Metals but Not Water. PLoS ONE, 11(8), e0160976.

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Quantifying Hydrogen Peroxide in Tissue

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Hydrogen peroxide (H₂O₂) generation in homogenates was measured using horseradish peroxidise/homovanillic acid (HRP/HVA) systems according to Ruch et al. (1983 (link)). In brief, 50 mg of tissue were homogenized with 2 mL of 1.15% KCl. Then, 10 μL homogenate was divided between two Eppendorf tubes. One tube received a reaction mixture for the calibration curve, which contained phosphate-buffered saline (PBS; pH 7.0) and HRP (1 U/mL) containing 400 μmol HVA. The second tube received PBS and 1 U/mL HRP.
Both tubes were incubated for 60 min at 37 °C. Then, PBS and 0.1 M glycine–NaOH buffer (pH 12.0) with 25 mM EDTA were added to each tube to stop the enzymatic reaction. Excitation was set at 312 nm and emission was measured at 420 nm (Perkin Elmer Luminescence Spectrometer, LS-50, Norwalk, CT, USA). Readings were converted into H₂O₂ concentration using the regression equation prepared from three series of calibration experiments with ten increasing H₂O₂ concentrations (range 10–1000 μM).

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