ODND probes were prepared by mixing approximately equimolar amounts of the corresponding complementary ODN dissolved in 25 mM Hepes, 1 mM MgCl2, 50 mM NaCl, pH 7.4. One of the complementary ODN used in these experiments contained the covalently linked fluorophore. The solutions were heated to 95°C for 5 min to dissociate intra-strand secondary structures and allowed to cool at room temperature to attain equilibrium. Fluorescence emission spectra of ODND or single-strand ODN (λem=500-650 nm, Cary Eclipse spectrofluorometer) were collected at a concentration of 2 µM by using λex=488 nm. The fluorescence intensity of ODNs and ODND was normalized by the concentration of Cy3 determined from the absorbance spectra (λmax=552 nm, ε552=150000 [M.cm]-1). Melting behavior was studied using a Hitachi U-2900 spectrophotometer equipped with a thermoelectric cell holder and a temperature controller. ODND were diluted in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 for melting experiments.
Eclipse spectrofluorometer
Manufactured by Agilent Technologies
Sourced in United States
The Eclipse spectrofluorometer is a laboratory instrument designed for fluorescence analysis. It measures the intensity of light emitted by a sample in response to excitation by light of a specific wavelength. The core function of the Eclipse spectrofluorometer is to provide accurate and reliable fluorescence data to support various analytical and research applications.
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Lab products found in correlation
U 2900 spectrophotometer, by Hitachi (1 mentions)
Cary 50 spectrometer, by Agilent Technologies (1 mentions)
Nitazoxanide, by Merck Group (1 mentions)
Infrared spectrophotometer, by Bruker (1 mentions)
Calf thymus dna ct dna, by Merck Group (1 mentions)
Rucl3 3h2o, by Merck Group (1 mentions)
Uv vis spectrometer, by Shimadzu (1 mentions)
Kaucl4, by Merck Group (1 mentions)
600 mhz spectrometer, by Bruker (1 mentions)
Sta 449 f1, by Netzsch (1 mentions)
Cary 300 spectrometer, by Agilent Technologies (1 mentions)
2400 series 2 analyzer, by PerkinElmer (1 mentions)
Inova 500 mhz spectrometer, by Agilent Technologies (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
Cary 300 uv spectrophotometer, by Agilent Technologies (1 mentions)
Matlab script, by MathWorks (1 mentions)
Lambda 950 spectrophotometer, by PerkinElmer (1 mentions)
Amx 600 spectrometer, by Bruker (1 mentions)
Cary 4000 spectrophotometer, by Agilent Technologies (1 mentions)
Fls920, by Edinburgh Instruments (1 mentions)
26 protocols using eclipse spectrofluorometer
1
Fluorescence-Based Thermal Denaturation of DNA Duplexes
2
GNP-siRNA Fluorescence Assay
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Fluorescence measurements with acridine orange were performed, as follows. Three different concentrations of GNPs were prepared: the stock solution, the stock solution diluted 10 times, and the stock solution diluted 100 times. 1 µL of acridine orange (0.5 µg/mL) was mixed with different volumes of these three GNPs solutions, namely, 1, 2, and 5 µL (additional sample with 10 µL was made for stock solution). The whole volume of probes was brought to 100 µL with water. These probes were used as reference values. For probes with siRNA, 1 µL of S and As siRNA solutions (1 µM) were added. All of the probes were left at room temperature for 30 min., and fluorescence was measured on Cary Eclipse spectrofluorometer at the excitation wavelength of 490 nm and emission wavelength of 525 nm.
3
Fluorescence Titration of RNA-Compound Interactions
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Fluorescence titrations were
carried out in 20 mM HEPES, 150 mM NaCl, using a Cary Eclipse spectrofluorometer.
All titrations started at a volume of 500 μL with the compound
at 1 μM. RNA was then titrated in from a high-concentration
stock (10 μM for HIV-1 FSS RNA; 40 μM for HIV-1 FSS DNA
and tRNA) in 1–10 μL increments. After RNA was added,
the solution was thoroughly mixed in the cuvette via pipetting and
allowed to stand for 10 min to reach equilibrium. Each measurement
was taken three times with a 1 min waiting period between scans to
confirm equilibrium was reached. Intensities were corrected for dilution.
carried out in 20 mM HEPES, 150 mM NaCl, using a Cary Eclipse spectrofluorometer.
All titrations started at a volume of 500 μL with the compound
at 1 μM. RNA was then titrated in from a high-concentration
stock (10 μM for HIV-1 FSS RNA; 40 μM for HIV-1 FSS DNA
and tRNA) in 1–10 μL increments. After RNA was added,
the solution was thoroughly mixed in the cuvette via pipetting and
allowed to stand for 10 min to reach equilibrium. Each measurement
was taken three times with a 1 min waiting period between scans to
confirm equilibrium was reached. Intensities were corrected for dilution.
4
Fluorescence Spectroscopy Procedure
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A Cary Eclipse spectrofluorometer with a xenon lamp and 5 mm slits was utilized. The synchronous mode was set at ∆λ = 60 nm and smoothing factor = 20. A Sonix IV model-SS101H 230 (USA) was utilized. A Consort pH meter was utilized for pH adjustment.
5
Absorption and Fluorescence Spectroscopy
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Electronic absorption spectra were recorded using a Cary 50 spectrometer. Room-temperature stationary fluorescence spectra were measured using a Horiba FluoroMax-4 spectrofluorometer, whereas temperature-dependent fluorescence spectra were recorded using a Cary Eclipse spectrofluorometer. The fluorescence spectra were corrected using a set secondary emissive standards.49 (link) Measurements at different temperatures were carried out using an Oxford OptistatDN cryostat.
6
Fluorescence Study of Lysozyme-BTS Interaction
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Fluorescence measurements were conducted on a Cary Eclipse spectrofluorometer. The excitation and emission splits were 5 nm. The synchronous fluorescence spectra were recorded by adjusting the data interval at 1 nm and the average time at 0.5 s. The measured range was 250–450 nm. Upon excitation at 280 nm, the fluorescence spectra of lysozyme-BTS systems were recorded at 298.15 K. Protein concentration was fixed at 0.02 mM and increasing concentrations of drug were added, from 0 mM to 0.20 mM. With the purpose of avoiding inaccurate results, inner filter effects were corrected for the quenching experiments by using the following expression: Fcorr = Fobs · e ^ [(Aexc + Aem)/2]; where Aexc and Aem are the absorptions of the systems at the excitation and the emission wavelength, and Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively. For data processing, UV-Vis-IR Spectral Software (FluorTools) was used [48 ].
7
Nitazoxanide and Metal Complexes Characterization
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Nitazoxanide with 99 % purity (NTZ) and the metal salts of RuCl3·3H2O, KAuCl4 and AgNO2 were purchased from Sigma Aldrich and used without further purification. Human serum albumin (HSA, A1887; globulin and fatty acid free) and calf thymus DNA (Ct-DNA) were used for the biological study and purchased from Sigma Aldrich.
Infrared spectra (IR) for the ligand and metal complexes were recorded on a Bruker infrared spectrophotometer in the range of 400–4000 cm−1. The molar conductance of the 10−3 M complex solution in DMF was measured on a HACH conductivity meter. A Bruker 600 MHz spectrometer was used to record the 1H NMR spectra in DMSO d6 solution. Electronic spectra of the metal complexes were obtained with a Shimadzu UV/Vis spectrometer in the range of 200–800, while fluorescence experiments were carried out on a Cary Eclipse spectrofluorometer from 300 to 600 nm. Thermogravimetric analysis TG-DTG experiments were conducted using a NETZSCH STA 449F1 thermal analysis system under air at a flow rate of 30 mL/min and a 10 °C/min heating rate for the temperature range of 25–800 °C, and the data were analyzed by Proteus software. The percentage of the metal ions was calculated thermogravimetrically as metal oxides.
Infrared spectra (IR) for the ligand and metal complexes were recorded on a Bruker infrared spectrophotometer in the range of 400–4000 cm−1. The molar conductance of the 10−3 M complex solution in DMF was measured on a HACH conductivity meter. A Bruker 600 MHz spectrometer was used to record the 1H NMR spectra in DMSO d6 solution. Electronic spectra of the metal complexes were obtained with a Shimadzu UV/Vis spectrometer in the range of 200–800, while fluorescence experiments were carried out on a Cary Eclipse spectrofluorometer from 300 to 600 nm. Thermogravimetric analysis TG-DTG experiments were conducted using a NETZSCH STA 449F1 thermal analysis system under air at a flow rate of 30 mL/min and a 10 °C/min heating rate for the temperature range of 25–800 °C, and the data were analyzed by Proteus software. The percentage of the metal ions was calculated thermogravimetrically as metal oxides.
8
Interaction of mithramycin A with DNA
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Samples were prepared in buffer solutions containing 10-mM PO43−, 50-mM NaCl and 10-mM MgCl2 (pH 7.4). The concentrations of mithramycin A (ϵ400 = 10 000 M−1 cm−1) and poly(dG-dC)2 (ϵ254 = 8400 M−1 cm−1) were determined spectrophotometrically. Poly(dG-dC)2 was incubated with TriplatinNC at a ratio of 0.05 drug/nucleotide at 37°C for 1 h. Varying amounts of mithramycin A were added in the r range of 0.0–0.25 to modified and untreated DNA. The samples were kept for 24 h at 37°C in the dark. The final concentration of poly(dG-dC)2 in the samples was 100 μM. Fluorescence spectra were recorded with a Cary Eclipse spectrofluorometer. To avoid photodegradation the fluorescence excitation wavelength was set to 470 nm. The absorbance of the samples at this wavelength was less than 0.05 with the inner filter effect therefore being neglected. Spectra were recorded in the range of 525–625 nm.
9
Luminescence Quantum Yield Determination
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UV–Vis spectra in dichloromethane solutions were recorded on an Agilent Cary 300 spectrometer (Santa Clara, CA, USA), for the fluorescence spectra, a Cary Eclipse spectrofluorometer has been used. All measured luminescence spectra were corrected for nonuniformity of detector spectral sensitivity. Rhodamine 6G (φfl 0.95) in ethanol was used as a reference for the luminescence quantum yield measurements. The luminescence quantum yields were calculated using equation:
where φi and φ0 are the luminescence quantum yields of the studied solution and the standard compound, respectively; Ai and A0 are the absorptions of the studied solution and the standard, respectively; Si and S0 are the areas underneath the curves of the luminescence spectra of the studied solution and the standard, respectively; and ni and n0 are the refractive indices of the solvents for the substance under study and the standard compound (ni 1.4242, DCM; n0 1.361, EtOH).
where φi and φ0 are the luminescence quantum yields of the studied solution and the standard compound, respectively; Ai and A0 are the absorptions of the studied solution and the standard, respectively; Si and S0 are the areas underneath the curves of the luminescence spectra of the studied solution and the standard, respectively; and ni and n0 are the refractive indices of the solvents for the substance under study and the standard compound (ni 1.4242, DCM; n0 1.361, EtOH).
10
Calibration of Carboxy-SNARF-1 in Mitochondria
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The calibration of carboxy-SNARF-1 in the bulk solution was done in two modes. In the first mode, the 20 μM solution of carboxy-SNARF-1 in the ‘respiration buffer’ was manually titrated in the pH range of 6.35‒10.41 by adding minute portions of concentrated HCl or NaOH and determining the resulting pH using a pH-meter equipped with a glass electrode. In the second mode, analogous experiment was performed in the presence of mitochondria suspension (OD600 = 0.3), in order to account for light scattering by the mitochondria. The fluorescence spectra were recorded on a Cary Eclipse spectrofluorometer in the 520–720 nm wavelength range, at a scan rate of 60 nm/min, following excitation at 488 nm, using 5 nm excitation slit and 10 nm emission slit of.
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