After equilibration for 60 minutes in HEPES buffer at 37°C, aortic segments were incubated for 45 minutes with the fluorescent probe 4,5-diaminofluorescein (2 µmol/L; Sigma-Aldrich; catalog No. D225). The medium was collected to measure baseline NO release, and the segments were incubated with phenylephrine (1 µmol/L, 5 minutes) and acetylcholine (10 µmol/L, 10 minutes). Thereafter, the medium was collected to measure agonist-induced NO release. Medium fluorescence was measured at room temperature using a spectrofluorimeter at an excitation wavelength of 492 nm and emission wavelength of 515 nm. To control for day-to-day fluorescence fluctuations, all experimental parameters were measured every day. NO release was calculated by subtracting baseline NO release from that evoked by acetylcholine. Blank samples were collected in the same way from segment-free medium to correct for background emission. The amount of NO released was expressed in arbitrary units/min·µg−1 protein. Variations in NO release were calculated as a percentage relative to controls.
4 5 diaminofluorescein
Manufactured by Merck Group
Sourced in United States, Germany
4,5-diaminofluorescein is a fluorescent dye used in laboratory applications. It is a derivative of fluorescein and emits green fluorescence upon excitation. The dye can be used as a detection and labeling agent in various biological and chemical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m2, by Molecular Devices (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Salicylic acid, by Merck Group (1 mentions)
Diethylenetriaminepentaacetic acid, by Merck Group (1 mentions)
4 aminoantipyrine, by Merck Group (1 mentions)
5 thio 2 nitrobenzoic acid, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Phenol, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Iron 2 sulfate heptahydrate, by Merck Group (1 mentions)
Spectrostar nano, by BMG Labtech (1 mentions)
4 protocols using 4 5 diaminofluorescein
1
Quantifying Acetylcholine-Induced Nitric Oxide Release
2
Nitric Oxide Synthase Assay for Hsp90 Activity
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A standard nitric-oxide synthase assay was used as an indicator of Hsp90 activity at 39.5 °C (47 (link)), wherein nitric-oxide synthase converts arginine into l -citrulline and nitric oxide (NO). The NO reacts with the amino groups in 4,5-diaminofluorescein (Sigma) and converts the substrate into fluorescent triazolofluorescein. Briefly, 8E5/LAV and ACH-2 cells were incubated up to 14 h with sampling every 2 h, and 105 cells were washed in PBS and seeded in black, clear bottom, 96-well plates in reaction buffer containing 100 μm l -arginine and 0.1 μm 4,5-diaminofluorescein and incubated for 5 min in the dark at room temperature. Fluorescence was measured at room temperature using a spectrofluorometer (SpectraMax M2, Molecular Devices) with an excitation wavelength at 495 nm and emission wavelength at 515 nm. The bandwidth was 10 nm for both excitation and emission, and the sensitivity was programmed to high. Thapsigargin was used at 10 μm as a positive control for nitric-oxide synthase activity.
3
Nitric Oxide Quantification Assay
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Supernatants were transferred into Costar 96-well black plate with clear bottom (MERCK, Germany) in duplicates. 4,5-Diaminofluorescein (Sigma-Aldrich, MO, U.S.A.) was added in the final concentration of 10 µM. Fluorescence was measured on Spectramax i3x multiplate reader (Molecular Devices, CA, U.S.A.) with excitation wavelength of 495nm and emission wavelength of 525nm. Values were corrected with control sample (pure media).
4
Fluorometric and Spectrophotometric Assay Protocols
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High-purity reagents and standards used for fluorometric and spectrophotometric assays including xanthine, xanthine oxidase from bovine milk, nitrotetrazolium blue chloride (NBT), horseradish peroxidase, hydrogen peroxide, 4-aminoantipyrine, phenol, iron (II) sulfate heptahydrate, salicylic acid, 5-thio-2-nitrobenzoic acid, 4,5-diaminofluorescein, Evans blue, sodium borohydride, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, QU dihydrate, QCT, RT, CHA, TX, and AA were from Sigma-Aldrich (Seelze, Germany/St. Louis, MO, USA), phosphate-buffered saline (PBS) was from Biomed (Lublin, Poland), sodium nitroprusside and NaOCl were from Avantor Performance Materials (Gliwice, Poland), while ECA, PB2, and PC1 were purchased from Phytolab (Vestenbergsgreuth, Germany). The species-specific flavonoids HRQ and PRQ were isolated earlier in our laboratory from the leaves of S. domestica [4 (link)]. All other chemicals were of analytical grade and obtained from Avantor Performance Materials (Gliwice, Poland). All activity studies were performed using 96-well plates and microplate readers: SPECTROstar Nano (BMG LabTech, Ortenberg, Germany) and Synergy HTX (BioTek, Winooski, VT, USA).
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