Methodology for measurement of the ethidium bromide excluded from bacteria has been previously published [13 ]. Briefly, cultures of P. aeruginosa were grown to logarithmic phase, pelleted, and resuspended at an OD of 0.7. Every 5 m following addition of ethidium bromide (5 μg/ml), 1 ml of culture was removed, pelleted, and the supernatant was analyzed using an excitation wavelength of 530 nm and detection wavelength of 585 nm. A control sample (containing EtBr but no bacteria) allowed normalization of the data and represents the maximal fluorescence intensity (measured as 420 nm and plotted as the ‘zero’ time for each sample). The measurements were performed on a Varian Cary Eclipse Fluorescence Spectrophotometer with a 20-nm excitation slit width. Three biological replicates were performed. For measurement of accumulation of ethidium bromide, P. aeruginosa was grown in CM9 to logarithmic phase (OD600 = 0.8) in the presence and absence of 300 μM each PUFA. Bacteria were pelleted, washed with PBS and resuspended in PBS at an OD600 = 0.4. After addition of 20 μM of ethidium bromide, fluorescence measurements were taken every 5 m for 1 h using a Varian Cary Eclipse Fluorescence Spectrophotometer with a 10-nm excitation slit width (excitation wavelength of 545 nm, detection wavelength of 600 nm). Two biological replicates were performed.
Cary eclipse fluorescence spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States, Australia, Italy, Germany, United Kingdom
The Cary Eclipse Fluorescence Spectrophotometer is a laboratory instrument designed to measure the fluorescence properties of samples. It is capable of performing excitation and emission scans, as well as time-based measurements. The instrument uses a xenon flash lamp as the light source and provides high-sensitivity detection and rapid scanning capabilities.
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Lab products found in correlation
Cary 60 uv vis spectrophotometer, by Agilent Technologies (10 mentions)
Cary 100 uv vis spectrophotometer, by Agilent Technologies (7 mentions)
Zetasizer nano z, by Malvern Panalytical (7 mentions)
Xenon flash lamp, by Agilent Technologies (6 mentions)
Escalab 250xi, by Thermo Fisher Scientific (6 mentions)
Cary 100 spectrophotometer, by Agilent Technologies (5 mentions)
Chemdoc xrs, by Bio-Rad (5 mentions)
Uv 1800 spectrophotometer, by Shimadzu (4 mentions)
Cary 500 spectrophotometer, by Agilent Technologies (4 mentions)
Microplate reader, by Agilent Technologies (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Prism 7, by GraphPad (4 mentions)
Cary 60 uv vis, by Agilent Technologies (3 mentions)
Quartz cuvette, by Hellma (3 mentions)
Lct premier xe spectrometer, by Waters Corporation (3 mentions)
Am 400 spectrometer, by Bruker (3 mentions)
V 650 spectrophotometer, by PerkinElmer (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Jem 2100 transmission electron microscope, by JEOL (3 mentions)
Uv 2450 uv vis spectrophotometer, by Shimadzu (3 mentions)
761 protocols using cary eclipse fluorescence spectrophotometer
1
Ethidium Bromide Exclusion Assay
2
Mitochondrial Function and ROS Evaluation
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The mitochondrial membrane potential (MMP, ∆Ψm) was examined via JC-1 staining (Beyotime, Jiangsu, China). Mitochondria (300 μg) in 2 ml respiration medium were incubated with JC-1 working solution at 37°C in the dark for 10 min, and then, mitochondrial respiratory function was initiated by a 15-μl mixture of 0.8 mol/L malic acid and 1 mol/L glutamic acid. The alteration of the fluorescence emission was detected using a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA, United States).
The mitochondrial ROS generation was measured fluorometrically using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma). Briefly, 300 μg of isolated mitochondria were incubated with fresh incubation medium containing 5 μM DCFH-DA at 37°C in the dark for 15 min. Fluorescence was determined for 2 min at 499 nm for excitation and 521 nm for emission using a Cary Eclipse fluorescence spectrophotometer (Varian).
The mitochondrial ROS generation was measured fluorometrically using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma). Briefly, 300 μg of isolated mitochondria were incubated with fresh incubation medium containing 5 μM DCFH-DA at 37°C in the dark for 15 min. Fluorescence was determined for 2 min at 499 nm for excitation and 521 nm for emission using a Cary Eclipse fluorescence spectrophotometer (Varian).
3
Quantifying Oxidative Stress in C. albicans
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C. albicans cells grown in liquid SD medium until the early (12 h) and late (24 h) exponential phases of cell growth were harvested, washed, and resuspended at 108 cells/ml in 100 mM sodium phosphate buffer (pH 6). The intracellular superoxide content was measured using dihydroethidium with 18.9 μM dihydroethidine [31 (link)]. Superoxide anions were measured using a Cary Eclipse fluorescence spectrophotometer (Varian) with λex = 518 nm, λem = 605 nm.
Cellular peroxide was estimated using 19.5 μM 2′,7′-dichlorofluoroscein diacetate (H2DCFDA) with a Cary Eclipse fluorescence spectrophotometer (Varian) with λex = 495 nm and λem = 525 nm, with previously described methods [12 (link)].
Cellular peroxide was estimated using 19.5 μM 2′,7′-dichlorofluoroscein diacetate (H2DCFDA) with a Cary Eclipse fluorescence spectrophotometer (Varian) with λex = 495 nm and λem = 525 nm, with previously described methods [12 (link)].
4
Dapoxyl Fluorescence Assay for Zika Virus
5
Fluorescence Characterization of Glyco-Dots
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The fluorescence measurements were carried out at room temperature using an Agilent Cary Eclipse fluorescence spectrophotometer.
Fluorescence quantum yields DMB1 or DMB2 (with a final concentration of 30 mm) was dissolved in aqueous 0.01 m PBS (pH 7.4). DMB1 and DMB2 (with a final concentration of 10 mm) with addition of peroxynitrite (with a final concentration of 100 mm) dissolved in aqueous 0.01 m PBS (pH 7.4). Rhodamine B (with a final concentration of 5 mm) was dissolved in aqueous 0.01 m PBS (pH 7.4). The UV/Vis absorbance measurements were carried out at room temperature using a Varian Cary 60 UV/Vis spectrophotometer. UV absorption values were collected at 470 nm. The fluorescence measurements were carried out at room temperature by an Agilent Cary Eclipse fluorescence spectrophotometer with an excitation wavelength of 480 nm. Integrated fluorescence curve between 500 nm and 850 nm. Finally, the fluorescence quantum yield was calculated according to Equation (1): Supramolecular self-assembly of glyco-dots DM probe (1 mm, DMSO) was added to a solution of TPE-based glycoclusters (15 mm, PBS buffer). The resulting mixture was stirred during 30 min to produce the supramolecular glyco-dots for subsequent experiments.
Fluorescence quantum yields DMB1 or DMB2 (with a final concentration of 30 mm) was dissolved in aqueous 0.01 m PBS (pH 7.4). DMB1 and DMB2 (with a final concentration of 10 mm) with addition of peroxynitrite (with a final concentration of 100 mm) dissolved in aqueous 0.01 m PBS (pH 7.4). Rhodamine B (with a final concentration of 5 mm) was dissolved in aqueous 0.01 m PBS (pH 7.4). The UV/Vis absorbance measurements were carried out at room temperature using a Varian Cary 60 UV/Vis spectrophotometer. UV absorption values were collected at 470 nm. The fluorescence measurements were carried out at room temperature by an Agilent Cary Eclipse fluorescence spectrophotometer with an excitation wavelength of 480 nm. Integrated fluorescence curve between 500 nm and 850 nm. Finally, the fluorescence quantum yield was calculated according to Equation (1): Supramolecular self-assembly of glyco-dots DM probe (1 mm, DMSO) was added to a solution of TPE-based glycoclusters (15 mm, PBS buffer). The resulting mixture was stirred during 30 min to produce the supramolecular glyco-dots for subsequent experiments.
6
Synthesis of Fluorescent Probe SSP5
7
Renilla Luciferase Fluorescence in Sorbitol
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Fluorescence spectroscopy studies of Renilla luciferase was studied in the presence of various final concentrations of sorbitol (0, 0.4 and 0.6 M) using a Cary-Eclipse fluorescence spectrophotometer equipped with a quartz cuvette.
Intrinsic fluorescence of Renilla luciferase (final concentration 5.9 μM) was determined with an excitation wavelength of 290 nm and emission spectra were recorded between 295 and 440 nm at 25 °C, similar to the description provided by Raeessi-Babaheydari et al. [23 ]. The excitation and emission slit widths of samples were kept at 5 nm. Additionally, the fluorescence spectra of 8-anilino-1-naphthalenesulfonic acid (ANS) were analyzed in the presence of various final concentrations of sorbitol (0, 0.4 and 0.8 M) using a Cary Eclipse fluorescence spectrophotometer. The concentration of Renilla luciferase used in the experiment was 6.7 μM, and the molar ratio of ANS to enzyme was 1:50. The ANS emission was observed within the range of 380–700 nm, while the excitation wavelength was set to 350 nm.
Intrinsic fluorescence of Renilla luciferase (final concentration 5.9 μM) was determined with an excitation wavelength of 290 nm and emission spectra were recorded between 295 and 440 nm at 25 °C, similar to the description provided by Raeessi-Babaheydari et al. [23 ]. The excitation and emission slit widths of samples were kept at 5 nm. Additionally, the fluorescence spectra of 8-anilino-1-naphthalenesulfonic acid (ANS) were analyzed in the presence of various final concentrations of sorbitol (0, 0.4 and 0.8 M) using a Cary Eclipse fluorescence spectrophotometer. The concentration of Renilla luciferase used in the experiment was 6.7 μM, and the molar ratio of ANS to enzyme was 1:50. The ANS emission was observed within the range of 380–700 nm, while the excitation wavelength was set to 350 nm.
8
DNA Binding Affinity Assay
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FDA was performed on Cary eclipse fluorescence spectrophotometer at a scanning speed of 600 nm/min. In direct displacement fluorescence assay, the protamine was titrated into a mixture of CTDNA and EtBr by exciting EtBr at 480 nm. Similar experiments were performed using DAPI as a probe by using excitation wavelength of 375 nm. In Reverse displacement assay, drug was titrated into already condensed DNA (protamine/DNA molar ratio of 0.1).
9
Ultrasound Impact on Tobacco Cell Viability
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Suspended tobacco cells were treated under various ultrasonic intensities of 160, 320 and 640 W, at 5, 8 and 10 min with 40 kHz frequency using a water bath ultrasound. Bath ultrasound (BANDELIN, sonorex digital 10p) at 40 kHz frequency was applied to tobacco cells to assess the cell viability against ultrasound waves in different ultrasonic intensities and times. Intracellular esterase activity was measured by the determination of fluorescein diacetate (FDA) concentration in the cells by Cary Eclipse Fluorescence Spectrophotometer. Application of continuous wave ultrasound was conducted for a total duration of either 5, 8, or 10 min and no pulsed ultrasound was applied (no duty cycle measurement) [43] (link). To measure the cell viability, the method described by Steward et al. was applied [16] (link). Briefly, samples containing tobacco cells treated with ultrasound under the various conditions were diluted (3×) with PBS (0.25 M, pH 8.75). After centrifuging (10,000×g, 15 min), 200 µL of supernatant was isolated and added to PBS containing fluorescein diacetate (FDA) solution (0.0125% w/w) with a mass ratio of 1:14. The suspension was incubated for 15 min at 35 °C. The samples were filtered through 0.45-µm Whatman filter to remove the cell debris. The absorbance of sample was measured at 490 nm using spectrofluorometry method and the samples cell viability was estimated.
10
Fluorescence Spectroscopy of Fibronectin
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The fluorescence studies were performed using Cary Eclipse fluorescence spectrophotometer. Titrations were performed by the addition of heparin to FN in the presence and absence of KI. The samples were excited at 295 nm and emission spectra were recorded in the range of 300–400 nm. The excitation and emission slits were set at 10 nm and scan rate was 600 nm min−1.
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