The spectroscopic and kinetic behavior of the reaction between sulfide and CysNO, GSNO and SNAP was followed by UV–visible spectroscopy in a FLUOstar Omega (BMG Labtech, Offenburg, Germany). Stock solutions (100 mM) were diluted 1:100 in 1 M TRIS pH 7.5, transferred to a UV-transparent 96-well plate (200 µl/well). The in-built automatic injector was filled with 50 mM Na2S solution for in-well titration experiments. Spectra (200–800 nm) were acquired before and every 2 s for 100 cycles after injection of 1–20 µl aliquots of Na2S. Spectra were analyzed using Omega data analysis software (BMG Labtech). All other studies were carried out in 3 ml-volume quartz cuvettes, kept at either 25.0 or 37.0±0.02 °C with continuous stirring (t2 peltier-type cuvette holder with TC1 temperature controller, Quantum Northwest, Liberty Lake, WA, USA) using a Cary 60 UV/vis spectrophotometer and analyzed using WinUV software (Agilent Technologies, Wokingham, Berkshire, UK). No differences in spectral changes were observed whether SNAP was incubated with sulfide in the presence or absence of DTPA (100 µM), indicating that transition metal contamination of our buffers was negligible.
Omega data analysis software
Manufactured by BMG Labtech
Sourced in Germany
Omega is a data analysis software developed by BMG Labtech. It is designed to analyze data generated from various laboratory equipment and experiments. The software provides functionalities for data processing, visualization, and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Winuv software, by Agilent Technologies (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Cary 60 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Celltiter glo luminiscent assay, by Promega (1 mentions)
Polarstar omega fluorimeter, by BMG Labtech (1 mentions)
Resazurin, by Merck Group (1 mentions)
4 protocols using omega data analysis software
1
Kinetics of Sulfide-Nitrosothiol Reactions
2
Lactate and ATP Quantification in Cultured Cells
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Cells were plated on 24-well microplates and cultured for 24 h. Medium was collected and lactate determined by using colorimetric lactate oxidase-peroxidase assay lactate kit (Spinreact, Spain) as instructed by the manufacturer. The concentration of lactate in the medium was calculated by comparison with the signal obtained from standard with known amount of lactate provided by the manufacturer. Signals determined in medium without cells were also analyzed and subtracted from the amount determined in the presence of cells. ATP levels in cells were determined by using the CellTiter-Glo Luminiscent assay (Promega, USA), following the instructions of the manufacturer. Briefly, the same amount of reactive solution was added to culture medium to produce cell lysis with gentle movement. The mixture was homogenized and transferred to a white polystyrene 96-well assay plate. A standard with known amounts of ATP were also added in the same plate and mixed with reactive solution in a 1:1 ratio. A POLAR Star Omega fluorimeter and Omega Data Analysis Software (BMG Labtech) was used to analyze luminiscence. ATP amount was referred to the total of cells counted by hemocytometer after trypsin detachment seeded in a 24 well plate seeded in parallel.
3
Urea and Urease Colorimetric Assays
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The urea content and urease activity were
both determined by colorimetric assays using the microplate reader
Fluostar Omega with Omega data analysis software (BMG Labtech, Ortenberg,
Germany).
both determined by colorimetric assays using the microplate reader
Fluostar Omega with Omega data analysis software (BMG Labtech, Ortenberg,
Germany).
4
Investigating Cell Viability Modulation
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H1299 cells were cultured in the presence or absence of 2 µg/mL DOX for 3 days. Three days after the addition of DOX cells were transfected with control siRNA or siRNA against β-catenin, GSK3α and/or GSK3β. Two days later, cells were seeded in triplicate in 96-well plates.
U2OS cells carrying a tetracycline-repressible (Tet-OFF) cassette for inducible cyclin E expression were cultured in media supplemented (endogenous cyclin E expression) or not (cyclin E overexpression) with 2 μg/mL DOX. Eight days after the removal of DOX (TET) the cells were transfected with control or BRCA1 siRNA and two days later seeded in triplicate in 96-well plates. U2OS cells carrying a tetracycline-inducible (Tet-ON) cassette for inducible cyclin D1 expression were cultured in media supplemented (cyclin D1 overexpression) or not (endogenous cyclin D1 expression) with 2 µg/mL DOX. Eight days after DOX addition, cells were transfected with control, BRCA1 or BRCA2 siRNAs and two days later seeded in triplicate in 96-well plates.
Viability was determined using resazurin-based assays at the indicated time points. Cells were incubated with 10 μg/mL resazurin (Sigma-Aldrich, R7017) in growth medium at 37°C for 2 h. Fluorescence was measured (544 nm excitation and 590 nm emission) using POLARstar Omega software and plate reader with data exported using Omega Data Analysis software (BMG Labtech).
U2OS cells carrying a tetracycline-repressible (Tet-OFF) cassette for inducible cyclin E expression were cultured in media supplemented (endogenous cyclin E expression) or not (cyclin E overexpression) with 2 μg/mL DOX. Eight days after the removal of DOX (TET) the cells were transfected with control or BRCA1 siRNA and two days later seeded in triplicate in 96-well plates. U2OS cells carrying a tetracycline-inducible (Tet-ON) cassette for inducible cyclin D1 expression were cultured in media supplemented (cyclin D1 overexpression) or not (endogenous cyclin D1 expression) with 2 µg/mL DOX. Eight days after DOX addition, cells were transfected with control, BRCA1 or BRCA2 siRNAs and two days later seeded in triplicate in 96-well plates.
Viability was determined using resazurin-based assays at the indicated time points. Cells were incubated with 10 μg/mL resazurin (Sigma-Aldrich, R7017) in growth medium at 37°C for 2 h. Fluorescence was measured (544 nm excitation and 590 nm emission) using POLARstar Omega software and plate reader with data exported using Omega Data Analysis software (BMG Labtech).
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