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    Detection Technical Notes

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    HydroFlex - Articles & Papers

    Reliable washing of Schott Nexterion® MTP-96 array plate with Tecan HydroFlex microplate washer

    Automated washing of magnetic Dynabeads® from LifeTechnologies with Tecan HydroFlex microplate washer

    In this article, we describe the use of Tecan’s HydroFlex™ washer equipped with a magnetic bead plate carrier for fast purification of a large number of samples using magnetic beads.In the protocol in our lab at the German Cancer Research Center (DKFZ), the affinity tag is Staphylococcus aureus protein A, which can be purified on immunoglobulins as the affinity matrix.

    VBC-Genomics in Vienna uses a Tecan HydroFlex™ system for automated washing of its microarrays for allergy testing. The arrays are spotted onto VBC’s proprietary surface, segmented into a 96 well format with a Whatman FAST® frame slide adapter and then processed by the HydroFlex, which halves the slide processing time and improves the signal quality andconsistency of results.

    Tecan’s new HydroFlex™ platform provides excellent performance, reliability and flexibility for a range of 96-well format applications, including washing of ELISAs, cellular assays andprotein arrays, and vacuum filtration-to-waste, such as for PCR clean-up. The platform’s outstanding on-line control features set new standards for plate washers.

    Infinite 200 PRO - Technical Notes

    Implementation on Tecan’s Infinite® M200 PRO multimode reader

    In recent years genetic reporter systems have greatly influenced analysis and understanding of gene expression, gene regulation and cellular responses in both eukaryotic and prokaryotic cells. A genetic reporter system consists of a promoter or a genetic element under analysis joined to a reporter gene in an expression vector. Expression of the reporter protein can be accomplished by measuring the protein itself or the enzymatic activity of the protein.

    ...

    Comparing luminescence measurements using the Photon-Counting Tube-based Infinite® M200 PRO with Photomultiplier Tube-based multimode readers

    Introduction

    Multimode microplate readers (MMRs) are commonly equipped with photomultiplier tubes (PMTs). PMTs are extremely sensitive detectors of light in the ultraviolet, visible and near-infrared ranges of the electromagnetic spectrum. A PMT multiplies the photocurrent produced by incident light by as much as 100 million times, enabling individual photons to be detected (1). PMTs in multimode readers are mainly used for fluorescence- and luminescence-based measurements, and can be run in two modes; photomultiplier tube mode (PMT mode) and photon-counting tube mode (PCT mode). This mode cannot be defined by the user, and is set at the manufacturing site.

    In PMT mode, which is commonly used for fluorescence measurements, the amplification rate of photocurrents – the ‘gain’ of the PMT – can be controlled by the user through the instrument’s software. A low gain value (low amplification) is used to measure high assay signals, whereas a high gain value (high amplification) is used to detect low assay signals.

    Virtually all MMRs on the market are also equipped with a feature to automatically determine the optimal gain level for an assay plate based on the highest signal on the plate. As the final data values are always related to the gain level applied, they are given as relative units (RFU, relative fluorescence units).

    For smaller photon fluxes, such as luminescence applications, the photomultiplier can be operated in PCT mode, where final data is given as absolute values in counts per second (cps). In this mode, the photomultiplier gain is set to a fixed level by the manufacturer so that a single photoelectron (resulting from a single photon incident on the primary surface) generates a very large current at the output circuit. This predefined gain setting guarantees maximum sensitivity, but the user cannot control the amplification rate, thus not preventing the detection of high signals which are outside the range of the PMT (‘OVER’ signals).

    ...

    Comparing absorbance measurements between the Quad4 Monochromators™-based Infinite® M200 PRO and a multimode reader using photodiode array technology

    Introduction

    The most widely established technology for UV-Vis absorbance measurement is a monochromator-based microplate reader. Spectrophotometers have undergone a great deal of development since their introduction in the early 1950s (1) and, in recent years, multimode readers using linear photodiode array (PDA) technology for absorbance measurements have become available. PDA-based readers incorporate an optical grating and a solid state array detector, enabling measurement of light intensity throughout the UV and visible regions of the spectrum. Similar to a monochromator, but much faster, they allow the entire UV-Vis spectrum of a sample to be captured within a few seconds per well. However, this technology suffers from a number of drawbacks, mainly due to high levels of stray light. This results in a dramatically limited dynamic measurement range (2). This technical note compares the results of basic absorbance measurements performed on an Infinite M200 PRO multimode reader equipped with Quad4 Monochromators with those from a multimode reader equipped with a PDA.

    ...

     

    Sensitivity uniformity of AlphaScreen measurements

     

    Introduction
    AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay) is a bead-based screening technology developed for fast, reliable and cost-effective detection of biological interactions. The AlphaScreen chemistry employs donor and acceptor beads that can be attached to various types of biologically relevant molecules. The  phthalocyanine photosensitizer molecules embedded in the AlphaScreen
    donor beads convert ambient oxygen into singlet oxygen when excited at 680 nm. The singlet oxygen molecules are able to cover a distance of up to 200 nm during their half-life of approximately 4 μs. If AlphaScreen acceptor beads are in close proximity to the donor beads, due to biological binding of their coupling partners, the singlet oxygen molecules are able to initiate a cascade of energy transfer steps in the acceptor beads, ultimately resulting in the generation of a strong light emission in the range of 520-620 nm. Due to the amplified signal, even small amounts of biological analytes can be detected [1].

    The Infinite F200 PRO is one of Tecan’s most reliable and sensitive filter-based multimode readers. It features all common measurement modes, including absorbance, fluorescence top/bottom, single and dual luminescence, fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET, including HTRF®). In addition, the Infinite F200 PRO is now capable of measuring AlphaScreen- and AlphaLISA-based assays.

    The Infinite F200 PRO uses its fluorescence module in combination with a dedicated dichroic mirror and optimized filter sets for AlphaScreen and AlphaLISA applications. While the excitation is performed at 680 nm in both cases, the emission filters are different for AlphaScreen and AlphaLISA readings; AlphaScreen signals are recorded using a 570 (100) nm filter, and AlphaLISA measurements use a 615 (20) nm
    filter that minimizes hemoglobin-caused assay background...

     

     

    Guidelines to recording excitation and emission spectra on the Infinite® M200 PRO and Infinite M1000 PRO

     Determination of instrument detection limits using the Glowell™ GLO-466 standard

     Enhanced Assay Performance using the Tecan Infinite® M200 Multimode Reader

    Enhanced Assay Performance using the Tecan Infinite® M200 PRO Multimode Reader

    Enhanced performance using the Tecan Infinite® M200 multimode reader

    Enhanced performance using the Tecan Infinite® M200 multimode reader

    Sensitive detection using the Tecan Infinite® M200 multimode reader

    Enhanced performance using the Tecan Infinite® M200 multimode reader

    Technical details and descriptions of applied features

    Optimizing the FI bottom sensitivity in the Infinite 200 PRO multimode reader series

    Optimizing the FI top sensitivity in the Infinite 200 PRO multimode reader series

    Optimizing the luminescence sensitivity in the Infinite 200 PRO multimode reader series

    Optimizing TRF sensitivity in the Infinite 200 PRO series multimode readers

    Based on the successful Infinite 200 series, Tecan has developed the Infinite 200 PRO, with new enhancements, like the automated adjustable z-focusing in the Infinite M200 PRO. The adjustable z-focus for FI top measurements, which offers equally high sensitivity for all plate formats, helping to implement assay miniaturization. It is a flexible tool to customize measurement parameters to plate type and assay volume Z-focusing can be performed either by using the automated zcalculation function in the fluorescence intensity measurement stripe or by selecting the z-position option of the instrument menu in i-controlTM software. The z-position dialog of the instrument menu of Infinite M200 PRO provides an additional function which allows performing z-position scans for both a signal and a blank well for each fluorescent top measurement label. Beside the optimal z-position of signal and blank, the zposition for the maximum signal to blank ratio is also displayed. This automated z-adjustment with integrated background value correction is particularly suited to cell-based applications using autofluorescent growth media, providing automatic optimization of the signal-to-background ratio. The desired z-positions can be selected and applied to the measurement script by the user. In this technical note the impact of z-focusing on the sensitivity of fluorescence top measurements on Infinite M200 PRO is presented.

    Implementation on Tecan’s Infinite® 200 Multimode Reader Series

    This Technical Note describes the successful
    implementation of Invitrogen’s LanthaScreen TR-FRET assay system on Tecan’s Infinite F200 filter-based multi modular detection system. The Infinite F200 has performed according to Invitrogen’s LanthaScreen certification program criteria and was successfully validated by Invitrogen as “LanthaScreen® Certified”.

    Cell proliferation and viability assays are commonly used to assess cell number and cytotoxic effects. In this study the performance of the Infinite M200 with respect to the MTT assay, CellTiter 96 AQueous One Solution Cell Proliferation Assay and CellTiter-Blue Cell Viability Assay, respectively, was evaluated.

    Scientists from Affymetrix® Inc., a world-leading supplier of microarray equipment and assays, evaluated the Tecan Infinite® 200 NanoQuant multimode microplate reader and two other spectrophotometers for DNA concentration analysis.
    In this note, data are presented from a comparison done by Affymetrix using Tecan’s Infinite 200 NanoQuant, Molecular Devices SpectraMax® Plus and the Nanodrop® ND1000 instrument.

    For measurement and differentiation of small amounts of RNA and DNA, two detection systems using fluorescent nucleic acid binding dyes have been tested - the Quant-iT PicoGreen® reagent to measure DNA and the RiboGreen® reagent for detection of small amounts of RNA.For detection and quantification of small amounts of dsDNA and RNA within a wide range of concentrations, the fluorescence intensity measurement with the Tecan Infinite® 200 series provides sensitive and accurate measurement results.

    Infinite M1000 PRO - Technical Notes

    Implementation on Tecan’s Infinite® M1000 PRO multimode reader

    Reporter Gene Assay
    In recent years genetic reporter systems have greatly influenced analysis and understanding of gene expression, gene regulation and cellular responses in both eukaryotic and prokaryotic cells. A genetic reporter system consists of a promoter or a genetic element under analysis joined to a reporter gene in an expression vector. Expression of the reporter protein can be accomplished by measuring the protein itself or the enzymatic activity of the protein...

    Guidelines to recording excitation and emission spectra on the Infinite® M200 PRO and Infinite M1000 PRO

    Guidelines to performing simultaneous fluorescence excitation and emission scanning (3D scanning) using the Infinite® M1000 PRO

    Determination of instrument detection limits using the Glowell™ GLO-466 standard

     Enhanced Assay Performance using the Tecan Infinite® M1000 Multimode Reader

    Z-optimization with ‘maximum signal to blank ratio’ feature improves fluorescence intensity top measurements on the Infinite® M1000 and Infinite F500

    Fluorescence-based DNA quantification in low volumes using the Infinite® M1000 and Infinite F500 multimode readers

    Luminescence scans on the Infinite® M1000 multimode reader

    This technical note describes the successful implementation of the new luminescence scan feature on the Infinite M1000 premium Quad4 Monochromators™-based multimode reader.

    Enhanced performance using the Tecan Infinite® M1000 multimode reader

    Enhanced performance using the Tecan Infinite® M1000 multimode reader

    Sensitive detection using the Tecan Infinite® M1000 multimode reader

    Enhanced performance using the Tecan Infinite® M1000 multimode reader

    Optimizing the FI bottom sensitivity in the Infinite M1000 multimode reader

    Optimizing the FI top sensitivity in the Infinite M1000 multimode reader

    Optimizing the TRF sensitivity in the Infinite M1000 multimode reader

    Optimizing the luminescence sensitivity in the Infinite M1000 multimode reader

    Implementation on Tecan’s Infinite® M1000 multimode reader

    Implementation on Tecan’s Infinite® M1000 multimode reader

    BRET1 and BRET2™ analysis on the Infinite® M1000: α2A adrenergic receptor mediated Gαi1 activation in HEK cells

    Implementation on Tecan’s Infinite® M1000 multimode reader

    This application note investigates how extended adjustable bandwidth settings maximize the sensitivity and reproducibility of the signal by fine-tuning the amount of excitation and emission light in a variety of fluorescence-based measurements.

     

    The Infinite® M1000 multimode microplate reader is the first monochromator-based instrument to date that meets the high level LanthaScreen® Certified Plus status. In this technical note we describe the instrument settings on the basis of experiments with the LanthaScreen® TR-FRET Control Kit on Tecan´s new Infinite® M1000 multifunctional detection system.

    Tecan´s Infinite® M1000 premium Quad4 Monochromators™ multimode microplate reader has been validated and certified by Cisbio Bioassays, France for compatibility with the HTRF® technology. In this technical note we describe the implementation and instrument settings on the basis of validation experiments conducted with the nfinite® M1000 and the HTRF® Reader Control Kit, HTRF® cAMP assay and HTRF® cytokine (TNFα) assay from Cisbio.

    Spark - Technical Notes

    In this technical note, we demonstrate the superior performance of the Spark reader compared to two other multimode readers from different providers. The detection limit for the far red fluorescent dye Alexa Fluor 647 – which has a fluorescence spectrum comparable to Cy5 – was determined, operating each instrument in MCR mode.

    The one-click Live Viewer software application turns the Spark microplate reader into a microscope. It enables easy and straightforward visual quality checks of cells in microplate wells or in Tecan's proprietary Cell Chip™, allowing researchers to automatically acquire and instantly view images of their cell samples immediately before the measurement is started.

    Cell-based assays are very popular in life sciences research, as they represent an in vitro system which offers results comparable to in vivo systems. However, this improved biological relevance is counterbalanced by the complexity of working with a living organism. Cell-based assays therefore require special precautions and care when planning and conducting experiments.

    This technical note describes the implementation of the DLR Assay System on Tecan’s next generation high-end multimode reader platform Spark. It can perform high sensitivity flash and glow luminescence measurements with a dynamic range of more than nine orders of magnitude. In addition, its enhanced luminescence module can measure multicolor assays with up to five different luciferases, as well as offering spectral scanning of luminescent substances.

    The Transcreener® ADP2 TR-FRET Red Assay is a competitive immunoassay for ADP with a far-red, time-resolved fluorescence-resonance-energy-transfer (TR-FRET) read-out. It is intended for the detection adenosine diphosphate (ADP) production by any kinase or ATPase, and has been developed specifically for high-throughput screening applications. The Spark was tested for its capacity to measure the Transcreener ADP2 TR-FRET Red Assay using the four different combinations of the Fusion Optics.

    The Transcreener ADP2 FP assay is a competitive FP assay based on the detection of ADP and is therefore compatible with any enzyme class that produces ADP, including protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases and glutamine synthetase. This technical note describes the instrument settings for the Transcreener ADP2 FP assay on Tecan’s Spark multimode microplate reader.

    The Transcreener ADP2 FI uses a simple fluorescence intensity read-out. It is a homogenous, competitive assay based on the detection of ADP with an extremely sensitive antibody against ADP. The Transcreener ADP2 FI assay is compatible with any enzyme class that produces ADP, including protein, lipid and carbohydrate kinases, ATPases, DNA helicases, carboxylases and glutamine synthetase. This technical note describes the instrument settings of Tecan’s Spark multimode reader for the Transcreener ADP2 FI assay.

    This technical note describes the optimized test set-ups, instrument settings and statistical equations that were used to determine the maximum instrument sensitivity of the Spark multimode reader for flash and glow luminescence.

    This technical note describes the optimized test set-ups, instrument settings and statistical equations that were used to determine the best possible detection limit for fluorescein in FI bottom mode.

    This technical note describes the optimized test set-ups, instrument settings and statistical equations that were used to determine the best possible detection limit for fluorescein in fluorescence intensity top reading mode (FI top).

    This technical note describes the optimized test set-ups, instrument settings and statistical equations that were used to determine the best possible detection limit of europium in time-resolved fluorescence intensity reading.

    With the Spark‘s luminescence module, users can perform high sensitivity flash and glow luminescence measurements. For optimized signal detection of very high and low signals within one experiment, optical density (OD) filters help to attenuate strong signals in the range from OD1 to OD3, thereby avoiding ‘over’ readings. This enables users to detect luminescence signals with a dynamic range of more than 9 orders of magnitude.Multicolor luminescence assays with up to five different luciferases, as well as luminescence spectral scanning (eg. of luminescent proteins) can be easily set - using an innovative filter-based approach, users can select any wavelength between 398 nm and 653 nm, with a step size of 15 nm and a 25 nm bandwidth.

    The new Spark multimode microplate reader is equipped with patent-pending High Speed Monochromators (HSM) enabling highly accurate, reproducible and ultra-fast absorbance measurements. This unique optical system offers an enhanced measurement range from 200 to 1,000 nm, ensuring optimal performance across the full range, particularly for absorbance measurements in the deep UV range, such as A260/A230 nucleic acid purity checks. The Spark reader is also suitable for measurements of low volume samples in both absorbance and fluorescence modes using the patented NanoQuant plate.

    This Technical Note describes the functions and benefits of the Spark multimode microplate reader and cell imaging module, as well as the SparkControl software and its dedicated Cell Counting and Cell Viability Application.

    This technical note compares the performance of the Spark’s cell counting module with (i) manual cell counting using C-Chip™ disposable hemocytometers and a standard microscope, (ii) a comparably priced, imaging-based cell counting device (Device A) and (iii) a high performance cell imager with counting capabilities (Device B).

    The new Spark reader is equipped with Tecan's unique Fusion Optics, which combine the benefits of filters and MCRs in a single multimode reader. The Spark can be equipped with filters, QuadX Monochromators or a combination of both using the ingenious Spark Fusion Optics. This allows users to independently choose between filters and MCRs for both excitation and emission independently from each other. So in addition to standard filter or MCR based measurements, it is now possible to measure with filter on excitation and MCR on emission site or vice versa. The new SparkControl™ software facilitates easy selection of filters or the MCR wavelength.