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Cellular Dopamine and Intracellular Calcium Signaling Using the Next Generation HTS Microplate Reader
BMG LABTECH
Two cell-based signaling assays from Invitrogen, Fluo-4 Direct™ Calcium Assay and Tango™-bla U2OS GPCR Assay, were performed on the PHERAstar FS HTS instrument. The PHERAstar FS has several unique features that enhance the performance of these assays including direct optic bottom reading, high resolution cell layer scanning, injection at the point of measurement and dual emission detection.
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MycoAlert Mycoplasma Detection Assay (Lonza) on the SpectraMax® L Microplate Luminometer
Molecular Devices
Mycoplasma infections are costly to the cell culturist as they change the morphology, viability and metabolism of the contaminated cells. Mycoplasmas are the smallest and simplest prokaryotes and depend entirely on their hosts for many nutrients due to their limited biosynthetic capabilities. Cell culture contamination by mycoplasma is difficult to detect as it does not produce pH changes or turbidity, and culture with antibiotics does little to deter this “invisible” menace.
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Quantitating Double-Stranded DNA with Quant-iT PicoGreen dsDNA Reagent and SpectraMax® Fluorescence Microplate Readers
Molecular Devices
Double-stranded DNA is typically quantitated in microplate readers by measuring the absorbance of the DNA solution at 260 nm. However, this method is only able to measure down to about 250 ng/mL on a typical absorbance microplate reader. For biological applications involving small samples, such as purification of DNA fragments for subcloning and quantitation of DNA amplification products, more sensitive methods are needed. The Quant-iT PicoGreen dsDNA Assay from Molecular Probes (Invitrogen) is
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Fluorescence-Based Measurement of Endothelial Cell Migration Through Collagen Using the BD Falcon FluoroBlok Insert System
Molecular Devices
Cell motility data from advanced time-lapse videomicroscopy and dynamic confocal reconstruction techniques of cell migration through 3-D extracellular matrices suggest that the inhibition of matrix-degrading enzymes by pharmacological inhibitors may induce a conversion in tumor cell invasion strategy from proteolytic invasion to non-proteolytic ameboid crawling.1 This observation raises the question whether proteolysis of the extracellular matrix is a critical parameter for invasion and metastas
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Automated Turnkey Solubility and Permeability Assays
Molecular Devices
Millipore Corporation and MDS Analytical Technologies have partnered to provide platforms for solubility and permeability screening. The objective is to create automation-compatible 96- and 384-based systems with pre-validated methods, devices and analysis that can be run manually or in conjunction with standard laboratory automation. The integration with analytical methods whose throughput, robustness, and sensitivity are compatible with the needs of ADME screening significantly improves overal
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High-Throughput Melamine Detection With SpectraMax® Absorbance Microplate Readers and Romer Labs’ AgraQuant Melamine Assays
Molecular Devices
The organic base melamine is used to make a number of products, including plastics, flame retardants, pigments, and fertilizers. The practice of adding melamine to animal feed and foods for human consumption in order to increase the apparent protein content has recently been reported. Because melamine contamination can cause serious illness or death in animals and people, there is increased interest in identifying methods for detecting melamine contamination in a variety of food products. In thi
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High-Throughput Melamine Detection with SpectraMax® Absorbance Microplate Readers and Abraxis’ Melamine ELISA Kit
Molecular Devices
The organic base melamine is used to make a number of products, including plastics, flame retardants, pigments, and fertilizers. The practice of adding melamine to animal feed and foods for human consumption in order to increase the apparent protein content has recently been reported. Because melamine can cause serious illness or death, there is increased interest in identifying methods for detecting melamine contamination in a variety of food products.
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Measurement of L-Malic Acid in Wines Using the SpectraMax® Plus384 Microplate Reader
Molecular Devices
Analysis of malic acid, residual sugar, volatile acidity and ammonia is very important for quality control during wine production. Enzymatic assays carried out in microplate format are quantitative and help achieve high throughput with respect to time and labor. These tests involved enzymatic conversion of the analytes to give NADH as a by-product. Measuring NADH production by measuring absorbance at 340 nm allows direct quantitation of the analytes in wine samples. Here we describe the use of M
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Measurement of Residual Sugar in Wine Using the SpectraMax® Plus384 Microplate Reader
Molecular Devices
Analysis of malic acid, residual sugar, volatile acidity and ammonia is very important in quality control during wine production. Enzymatic assays carried out in microplate format are quantitative and help achieve high throughput with respect totime and labor. The residual sugar assay involves enzymatic conversion of the analytes to give NADPH as a by-product. Measuring NADPH production by measuring absorbance at 340 nm allows direct quantitation of the analytes in wine samples.
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Measurement of Phenolic Compounds in Red Wines Using the SpectraMax® Plus384 Microplate Reader
Molecular Devices
Measurement of tannin, iron-reactive phenolics, anthocyanin and polymeric pigment in red wine is an important part of quality control in the wine industry. Precise and reliable measurement of phenolic compounds in wine is critical for making decisions during fermentation, maceration, pressing and blending. Harbertson et al. developed a comprehensive red wine phenolics assay in 2003.1 Traditionally this assay is performed using a cuvette-based UV-vis spectrophotometer. It is a time-consuming and
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ß-Lactamase-Inhibitor Protein Binding Affinities: Detection of Hydrolysis of ß-Lactam Substrate by TEM-1 Enzyme and Inhibitor Binding Using The FlexStation® 3 Microplate Reader
Molecular Devices
Bacterial resistance to ß-lactam drugs through the production of class A ß-lactamase enzymes is an increasing concern in the medical community.1 ß-lactamase inhibitory protein (BLIP) produced by the soil bacterium Streptomyces clavuligeris inhibits a number of class A ß-lactamases with a wide range of affinities, thereby restoring the effectiveness of life-saving antibiotics.2 BLIP’s known binding partners include Escherichia coli TEM-1, Serratia marcescens SME-1, Bacillus anthracis BlaI (nanomo
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Comparison of Photina Luminescent Calcium Mobilization Assays on both the FlexStation® 3 and FLIPRTETRA® Systems
Molecular Devices
G-protein coupled receptors (GPCRs) represent one of the most important therapeutic targets in drug discovery research. GPCRs are membranelocalized proteins that play an important role in cell signaling. When a receptor is activated by a ligand, the conformation of the receptor is modified, activating G-proteins inside the cell. An active G-protein has the potential to induce various cascades of intracellular messengers, including calcium.
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Image-based Quantification of Calcium Flux using the Opera™
This High Content Analysis application is useful for image-based quantification of drug induced GPCR activation. The Acapella™ Kinetic Intensity Analysis script used here can determine subpopulations of cells based on their calcium response. In pharmacological analysis this approach will decrease the number of false positives identified compared to systems that average the response across the entire population within a well.
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Image-based Quantification of the Huntingtin Protein using the Operetta
PerkinElmer
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The Transcreener® ADP2 FI Assay performed on PHERAstar and Omega microplate readers
BMG LABTECH
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Functional Screening of recombinant CHO-M1 cells using the PHERAstar from BMG LABTECH
BMG LABTECH
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Flexible Systems for High Throughput Screening
HighRes Biosolutions
High Throughput Screening (HTS) labs are required to screen ever-increasing numbers of compounds against an increasing number and variety of assay types. With a level of flexibility and adaptability unsurpassed in the world of automated systems, HighRes’ MicroStar™ is ideal. It meets not only today’s lab requirements; it also anticipates the easy exchange of today’s components with tomorrow’s technology.
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A Leukocyte Adhesion Assay Performed on BMG LABTECH’s FLUOstar OPTIMA
BMG LABTECH
Gene regulatory networks were used to identify novel regulatory hubs genes involved in the inflammatory response in human umbilical endothelial cells (HUVECs). To assess the potential role of these genes a leukocyte adhesion assay was utilized to screen for potential inflammatory markers. The FLUOstar OPTIMA microplate reader from BMG LABTECH was used for cell based bottom optic measurements and well scanning. The results show good reproducibility and can be adapted to e.g. primary neutrophils.
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Tag-liteTM: New HTRF® cellular platform for cell surface receptors’ study and screening
Cisbio Bioassays
In a recent publication, Maurel et al* presented the combination of Cisbio’s HTRF and Covalys’ SNAP-and CLIP- Tag technologies to investigate cell-surface protein-protein interaction. In this study, an application for GPCR oligomerization was accurately documented and the advantages of this new approach presented.
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Size-selective assessment of tight junction paracellular permeability using fluorescently labelled dextrans
BMG LABTECH
A method for the quick and reliable analysis of size-selective paracellular tracer diffusion using fluorescent dextrans of different molecular weights is described. The assay could be easily adapted to large scale screens to identify genome-wide regulatory pathways or small molecules to modify junctional permeability. Using BMG LABTECH´s multimode plate reader FLUOstar OPTIMA for these experiments allows for quick and consistent fluorescence measurements in either kinetic or endpoint format.
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Detection of NADH and NADPH with the Omega’s High Speed, Full UV/Vis Absorbance Spectrometer
BMG LABTECH
NAD+, NADH as well as NADP+, NADPH have been known to play vital roles in energy metabolism, antioxidation, and reductive biosynthesis. Changes in these systems can be monitored using absorbance spectroscopy. NADH and NADPH absorb light at 340 nm, whereas NAD+ and NADP+ do not. The FLUOstar, POLARstar and SPECTROstar Omega microplate readers from BMG LABTECH all have a UV/Vis spectrometer that can measure any absorbance range from 220-850 nm.
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Measuring Mitochondrial Membrane Potential using the FLUOstar Omega Microplate Reader
BMG LABTECH
Measurement of the mitochondrial membrane potential is useful in a variety of research areas. Mitochondrial membrane potential is usually measured using non-invasive cationic dyes. These dyes are sequestered into the mitochondrial matrix in amounts directly proportional to the membrane potential. BMG LABTECH’s FLUOstar Omega was used in an hts assay measuring mitochondrial membrane potential in human cells. The results presented here were gained using primary human fibroblast cells from controls
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Evaluation of PARP Inhibitors: Performed on BMG LABTECH’s FLUOstar Omega
BMG LABTECH
Poly (ADP-ribose) polymerases (PARP) transfer ADP-ribose to itself and other nuclear proteins such as histones, therefore PARP play a crucial role in regulating DNA repair. A luminescent cellular PARP assay and a fluorescent cell viability assay were performed on BMG LABTECH’s FLUOstar Omega to evaluate PARP inhibitors. PARP inhibition has been demonstrated to potentiate the cytotoxicity of anti-cancer drugs and ionising radiation. The ability of the FLUOstar Omega to measure absorbance, lumines
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Nephelometric Monitoring Growth of Candida albicans Using BMG LABTECH’s NEPHELOstar
BMG LABTECH
Laser nephelometry has been shown to be a reliable technique for the measurement of drug solubility in 96-well plate format. In this note the use of BMG LABTECH´s NEPHELOstar to investigate the effects of complexation on the drug antimycotic activity is described. Laser nephelometry can distinguish between the concentration at which the drug just goes into or just comes out of solution. This is presented in phase solubility studies. Furthermore nephelometry was used to monitor fungal growth.
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New Transcreener® ADP2 FP Assay performed on BMG LABTECH’s PHERAstar Plus HTS Microplate Reader
BMG LABTECH
Transcreener™ ADP2 Assay kits are far-red competitive fluorescence polarization immunoassays based on the detection of ADP. Any enzymatic reaction that uses ATP in a range from 0.1-1000 µM can be monitored.
With its dual emission detection and five photomultiplier tubes, BMG LABTECH´s PHERAstar Plus provides the speed and sensitivity needed to take full advantage of BellBrook Labs Transcreener™ technology. Furthermore, a specific optical module was developed, thereby making assay setup simple.
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Use of Nucleofector® Technology to Establish Stably Expressing Cell Lines
Amaxa GMBH
The number of stably expressing clones resulting from standard transfection experiments is often quite low, especially for cells which are difficult to transfect. This limits many research applications. We show here that the Nucleofector® Technology can successfully be used to generate stably expressing clones at high efficiency. Although a general protocol cannot be given, the data shown here can serve as a guideline for fast establishment of stable transfection protocols for various cell lines
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The Nucleofector® 96-well Shuttle® System – Nucleofection® of Cell Lines and Primary Cells in an Automated Framework
Amaxa GMBH
Screening of siRNA or cDNA libraries requires an automated high-throughput transfection process. Besides mere throughput considerations, the main drivers for automation are the standardization and robustness of the process. Stability of all cell parameters is the key to statistically relevant data and valid results. Therefore we analyzed the stability of transfection results of different cell types during a time span typically needed to screen a focused siRNA library using Nucleofection®. The tr
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High-Throughput Nucleofection® of Primary Rat Hippocampal Neurons
Amaxa GMBH
Postmitotic neurons are routinely difficult to transfect. This application note describes the efficient, high-throughput Transfection® of plasmid DNA into primary hippocampal neurons. Using amaxa’s Nucleofector® 96-well Shuttle® System, 30 to 50 % of neurons were successfully transfected and showed normal neuronal development. In addition, quantitative down-regulation of target proteins by RNA interference was achieved.
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Nucleofection® – High Transfection Performance and Superior Preservation of Functionality
Amaxa GMBH
Nucleofection® has become the method of choice whenever transfection of primary cells or hard-totransfect cell lines is required. In this application note we show that Nucleofection® of frequently used primary cells results in highly efficient transfer of DNA and other substrates while at the same time maintaining excellent cell viability and post transfection functionality.
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First siRNA Library Screening in Hard-to-Transfect HUVEC and Jurkat Cells
Amaxa GMBH
High-throughput transfection of siRNA libraries has become a valuable tool in target identification and validation. However, such screenings have so far been constrained to mostly easy- to-transfect adherent cell lines. amaxa’s Nucleofector® 96-well Shuttle® System extends these approaches to primary and hard-to-transfect cells. In this application note we report results of two different screening approaches in hard-to-transfect cell types using Thermo Scientific Dharmacon siARRAY® siRNA librari
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High Speed FRET based SNP Genotyping Measurement on the PHERAstar
BMG LABTECH, KBiosciences
Single Nucleotide Polymorphisms (SNP`s) have become an invaluable tool in the field of Genetic Research. Here we show the use of BMG LABTECH´s PHERAstar multimode HTS plate reader for high speed FRET based SNP Genotyping measurement. The new KASPar™ system, developed by KBiosciences, was used to assess the best performance of the PHERAstar. Optimized optical modules with dual emission lead to improvements in speed and accuracy of measurement.
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Molecular Probes® NanoOrange® Assay Performed on BMG LABTECH FLUOstar OPTIMA Microplate Reader
BMG LABTECH
The fluorescent NanoOrange® Protein Quantitation Kit from Molecular Probes® (Invitrogen) can be easily adapted for use with a microplate reader such as the FLUOstar OPTIMA from BMG LABTECH and used in a high throughput manner. High and small protein concentration range is evaluated. NanoOrange® can also be performed on other BMG LABTECH microplate readers, including the FLUOstar and POLARstar Omegas, as well as the NOVOstar and PHERAstar.
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SUMO FRET-based assays performed on BMG LABTECH’s NOVOstar
BMG LABTECH
The small ubiquitin-like modifiers SUMO1 and SUMO2 and their processing by the protease SenP1 was investigated by using an in vitro FRET assay performed on BMG LABTECH´s NOVOstar. The described assay has applications in SUMO protease characterization, enzyme kinetic analysis and high-throughput inhibitor screening.
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Investigation of the stereoselectivity of an anti-amino acid antibody utilizing tryptophan fluorescence
BMG LABTECH
The binding sites of proteins such as antibodies are known to often
contain tryptophan (Trp) residues, whose fluorescent properties may be
altered upon ligand binding. Conformational changes within the binding
site or simply the presence of the ligand can result in either fluorescence
quenching or enhancement, which may be utilized to quantitatively
investigate protein-ligand interactions.
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A New Matrix Application Device for MALDI Tissue Imaging
Bruker Daltonics
ImagePrep™ provides highly reproducible matrix preparations for MALDI tissue imaging in a fully automated process.
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Cisbio´s HTRF® Cortisol Assay
BMG LABTECH and Cisbio International
Cortisol is a corticosteroid hormone present in many metabolic processes, inducing key enzymes of the whole metabolism. Cisbio´s HTRF® cortisol assay allows fast and efficient determination of cortisol in complex samples such as serum and whole cells. In this poster we describe the performance of the cortisol assay on BMG LABTECH´s PHERAstar. With its optimized HTRF® optical module the PHERAstar is the ideal tool to run HTRF® assays.
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Analysis of Prostate Tumour Cell Invasion Using BD FluoroBlok and FLUOstar OPTIMA
BMG LABTECH and Paterson Institute for Cancer Research, University of Manchester
The FLUOstar OPTIMA is used for reading the BD Biosciences FluoroBlok™ tumour cell invasion system. In this poster we show that the results are comparable to the traditional invasion assays. The introduced method is fast, saving time and labour. The FLUOstar OPTIMA is able to take multiple readings over time at a user defined temperature.
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HTRF® IP-One Assay Performed on the PHERAstar and RUBYstar Plate Readers
BMG LABTECH and Cisbio International
BMG LABTECH is a leader in microplate reader technology. Cisbio’s HTRF IP-ONE screening assay for the Gq coupled GPCR pathway has been applied on two of BMG LABTECH´s HTS readers. One reader is the RUBYstar, a dedicated time-resolved fluorescence (TRF) HTS machine; the other is the PHERAstar, a multifunctional HTS machine that can read in all detection modes (fluorescence intensity, TRF, fluorescence polarization, luminescence, and absorbance).
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Invitrogen’s LanthaScreen™ TR-FRET Tyrosine Kinase Assay Using the PHERAstar
Invitrogen Corporation and BMG LABTECH
Several tyrosine kinases were characterized using Invitrogen´s LanthaScreen™ assay. The LanthaScreen™ TR-FRET platform combines terbium as the donor group and fluorescein as the acceptor species.BMG LABTECH’s PHERAstar microplate reader provides the ideal platform to simplify assay development. With its dual wavelength emission detection and five photomultiplier tubes, the PHERAstar provides the speed and sensitivity needed to take full advantage of Invitrogen’s LanthaScreen™ technology.
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Invitrogen’s LanthaScreen™ TR-FRET Protein Kinase C Assay Using the PHERAstar
Invitrogen Corporation and BMG LABTECH
BMG LABTECH’s PHERAstar microplate reader provides the ideal platform to simplify assay development. With its dual wavelength emission detection and five photomultiplier tubes, the PHERAstar provides the speed and sensitivity needed to take full advantage of Invitrogen’s LanthaScreen™ technology.
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Promega’s Multiplexed Cell Viability and Apoptosis Assays Performed on the PHERAstar
Promega Corporation and BMG LABTECH
Today’s high-throughput screening facilities face increasing demands to generate more information from their existing compound libraries. In this application note, we demonstrate the combination of several Promega cell-based assays (CellTiter-Blue®, Apo-One® and Caspase-Glo® 3/7) multiplexed in both low-volume 384 and 1536-well plate formats. The BMG LABTECH PHERAstar microplate reader is used to record both luminescence and fluorescence, depending on the multiplex combination.
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Promega’s Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar
BMG LABTECH and Promega Corporation
By multiplexing a reporter assay (EnduRen™, ViviRen™) with a cell viability assay (CellTiter-Glo®), it is possible to determine if reporter response variations are due to changes in cell number and health. In this poster, we demonstrate the combination of several Promega cell-based assays multiplexed in both low-volume 384- and 1536-well plate formats using the BMG LABTECH PHERAstar microplate reader to record luminescence.
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Molecular Probes PicoGreen® Assay Performed on BMG LABTECH POLARstar OPTIMA Microplate Reader
BMG LABTECH
The PicoGreen® dsDNA quantitation reagent from Molecular Probes is a highly sensitive fluorescence assay for dsDNA detection. Using BMG LABTECH´s POLARstar OPTIMA for measuring this quantitation assay a linear range of more than four magnitudes is obtained. The limit of detection was calculated to be 21 pg dsDNA / 200 µl showing that the POLARstar OPTIMA meets all requirements needed for a high performance.
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Simultaneous Dual Emission Detection for Fast Kinetic BRET Assays
BMG LABTECH
Bioluminescence Resonance Energy Transfer (BRET) is a system for monitoring intermolecular interactions in vivo. The BRET2™ demo kit has been used to prove the feasibility of performing a BRET assay on the POLARstar OPTIMA microplate reader. The POLARstar OPTIMA’s internal reagent injectors for 384-well plate format combined with high-end simultaneous dual emission detection offer a unique advantage for fast kinetic assays where simultaneous emission detection at two wavelengths is required.
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ORAC Assay on the FLUOstar OPTIMA to Determine Antioxidant Capacity
BMG LABTECH
BMG LABTECH has performed and optimized the fluorescence-based Oxygen Radical Absorbance Capacity (ORAC) assay on the multifunctional FLUOstar Optima microplate reader. The ORAC assay can be measured both directly and indirectly. The FLUOstar Optima has the ability to read in three modes (absorbance, luminescence, and fluorescence), has a standard 45°C incubation chamber, and has optional onboard injectors; thus making it the ideal machine to perform the ORAC assay.
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Detection of Tyrosine Kinase Activity using the PHERAstar in AlphaScreen™ mode
BMG Labtech
Tyrosine kinases are important regulators in cellular processes. Kinases have been found to be involved in diseases such as cancer and cardiovascular, therefore, molecules that modulate kinase function are expected to be promising new drug targets. In this application note, we describe the performance of an AlphaScreen™ tyrosine kinase assay on BMG LABTECH´s PHERAstar using an AlphaScreen™ specific excitation LASER.
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Assay Optimization and Profiling in uHTS Format
Deerac Fluidics,Promega Corp
We demonstrate the use of Promega’s Kinase-Glo® Plus Luminescent Kinase Assay for miniaturized kinase assays using Deerac Fluidics’ Equator™ HTS liquid handling system. The Kinase-Glo® Plus Assay is a homogeneous method of measuring kinase activity by quantifying ATP in solution following a kinase reaction. The Equator™ HTS is a noncontact liquid dispenser capable of delivering volumes from 20µl down to 50nl.
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Keeping DMSO Concentration Below 0.5% to Minimize its Effect in HTS Assays
Deerac Fluidics,Labcyte
The Labcyte Echo™ 550 liquid handler utilizes acoustic drop ejection (ADE) to transfer 2.5-250 nL of compounds in DMSO directly from storage plates to assay plates. Deerac Fluidics Latitude™ bulk dispenser, which uses “spot-on” technology, can precisely deliver as low as 50 nL. The Latitude can be used to rapidly add pure DMSO to specific wells so that all assay wells have the same DMSO concentration.
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Automated Dispensation of Yttrium Oxide SPA Imaging Beads, into 1536-Well Plates Using Deerac Fluidics’ Equator™ HTS Pipetting System
Deerac Fluidics
Here we demonstrate the successful combination of the use of the Equator HTS pipetting system to dispense Streptavidin coupled Yttrium Oxide (YOx) SPA Imaging beads. 2ul of YOx beads were dispensed into1536-well plates containing an excess of 3H-biotin. Plates were read on LEADseeker™ Multimodality Imaging System and analyzed for accuracy and reproducibility. CVs of 6-7% were obtained. Outliers were within spec of 10 or less per 1000 wells.
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