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    Detection Application Notes

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    GENios - Applications Notes

    We demonstrate here an example for the quantification of DNA, comparing absorbance (260 nm) with fluorescence detection using PicoGreenTM. We are able to show the advantage to have instrumentation available for both detection techniques, dependent on the yield of extracted DNA. The DNA samples used for the measurements were plasmid DNA and genomic DNA, extracted with the TECAN GENESIS RSP 150.

    In this application note we describe the use of a TECAN GENios multifunctional microplate reader for spectrophotometric measurements of CY5. Absorbance scans of CY3 and CY5 have been performed using a TECAN SAFIRE monochromator based microplate reader.

    GENios Plus - Application Notes

    The present assay exemplifies a rapid and convenient way of monitoring H2O2 release associated with respiratory burst in freshly isolated or cultured leukocytes. The assay is microplatebased and utilizes the fluorescence generated from the administered HVA. The fluorescence signal can be detected with the SPECTRAFLUOR Plus microplate-fluorometer. The protocol is applicable to basic research as well as to clinically-related analyses of cells from diseased individuals.

    Three anchorage-dependent leiomyosarcoma cell lines, and one suspension-growing Burkitt’s type non-cleaved B lymphoma line, Sc-1, were selected for the experiments. Cells were labelled with the various lipophilic dyes as described in the Application Note treating DiI labelling, or with 2 μM of Calcein AM or the CellTracker. For direct comparisons of the potential release of the various lipophilic dyes, labelled cells were allowed to grown for various time intervalls. Fluorescence present within the cells and released into the medium, was measured with the SPECTRAFLUOR Plus.

    The aim of all the various studies described in our application notes is to demonstrate the applicability of our microplate fluorometers (SPECTRAFLUOR or SPECTRAFLUOR Plus) for investigations using cell cultures including growth/ proliferation tests, cytotoxicity studies, studies on cell adhesion etc.

    The majority of luciferase reporter gene assays in vitro using luminometers are somewhat limiting for studies to examine the mode of gene regulation over time within the same cell population. We show here an application on the luciferase reporter gene system to monitor promoter activity comparatively, after transfection into a number of established and primary cells. This type of quantitation of cellular gene activity was possible using the multifunctional SPECTRAFLUOR Plus.

    GENios Pro - Application Notes

    This application note deals with flash luminescence measurements performed on Tecan’s multifunctional injector plate reader GENios Pro by using a CHO mito-PhotinaTM based receptor-ligand assay.

    Ion channels are important drug targets because of their critical role in nerve, cardiac, endocrine, and skeletal muscle tissues.This Application Note demonstrates the use of Tecan's GENios Pro instrument as a suitable platform for development of VSP ion channel assays in both pharmaceutical and academic environments. We demonstrate the portability of an assay developed on the GENios Pro to an ion channel HTS platform, the VIPR® (Aurora Discovery), by comparing data obtained from both instruments.

    The current technical note introduces two applications implemented and verified on the GENios Pro, one of Tecan’s multifunctional microplate readers. These applications utilise HTRF® (Homogeneous Time-Resolved Fluorescence) technique to generate the assay readout (CIS bio international, France).

    This technical note introduces the ULTRA Evolution and the GENios Pro, both multifunctional plates readers of Tecan, for dual color luminescence measurement. The Chroma-Luc® Technology, a homogenous dual reporter gene assay from Promega (US), was successfully evaluated using lysates containing the Chroma-Luc® luciferases as demo systems.

    JC-1 is a fluorescence mitochondrial potential sensor, which is mainly used for FACS analysis for detection of mitochondrial depolarization occurring during the early stages of apoptosis. This technical note shows that JC-1 could also be used in a microplate fluorometer for a basic differentiation between live and dead cells. Using the bottom reading option, Tecan Ultra Evolution, Safire and GENios Pro are capable of detecting the effects of Antimycin on cells and distinguish between live and dead cells.

    The data clearly show the ability of Tecan Ultra Evolution, Safire and GENios Pro to detect the two membrane permeant dyes, Calcein and Hoechst 33342, with the bottom reading option of the instruments.

    Hypericin is a powerful, naturally occurring photosensitizer that is found in Hypericum perforatum plants, commonly known as St. John's wort.The data clearly show the ability of Tecan Ultra Evolution, Safire and GENios Pro to detect the photosensitizer Hypericin with the bottom reading option of the instruments.

    Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequorea victoria, or from the sea pansy, Renilla reniformis.The purpose of this technical note was:a) to determine the optimal filters for eGFP b) to compare the efficiency of a top and bottom measurement c) to compare Costar and Greiner plates d) to determine the ‘detection limit’ for transfection studies. All measurements were performed with Tecan Ultra Evolution, Safire and GENios Pro.

    HydroFlex - Application Notes


    Introduction

    The enzyme-linked immunosorbent spot (ELISpot) assay is a very sensitive immunoassay, allowing detection of secreted cytokines at the single cell level. Due to its high sensitivity, the ELISpot assay is used to study small populations of active cells, such as those found in specific immune responses. The interferon gamma (IFNγ) ELISpot assay has been widely used to investigate specific immune responses in various diseases, including infections and cancer. Depending on the cytokine analyzed, the ELISpot assay can also be used to discriminate between different subsets of activated T-cells, such as T-helper (Th) 1 IFNγ-producing and Th2 IL10- producing cell types...

     

    In this application note we describe the use of Tecan’s HydroFlex™ washer equipped with the smart-2 MBS magnetic carrier for automated washing of Dynabeads® in an ELISA. Using Dynabeads® in combination with the HydroFlex™ plate washer confi gured for magnetic bead washing, it is possible to run bead-based ELISA with the convenience of the 96-well plate format and the ease of handling known from traditional well-based ELISA.

    In this application note we describe the use of Tecan’s HydroFlexTM washer equipped with a magnetic bead plate carrier for fast purification of a large number of samples using magnetic beads.For the detection of the luminescence signal Tecan’s Infinite® F200 multimode reader was used.

    This technical note describes how the Tecan HydroFlex™ platform was successfully evaluated for gentle washing of strongly adherent cell-line A431 as well as for the weakly adherent P815 cells in a 96-well format. For the cell-based assay described in this technical note, a HydroFlex platform, equipped with a standard wash head suitable for ELISA and cell washing, was used.

    This technical note describes the purification of PCR products via automated vacuum filtration using the modular Tecan HydroFlex™ platform equipped with the vacuum filtration option.

    HydroSpeed - Application Notes

    using Tecan’s HydroSpeed™ plate washer for EMD Millipore’s MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel

    using Tecan’s HydroSpeed™ plate washer with Cell Protection™ wash settings

    using Tecan’s HydroSpeed™ plate washer and Infinite® F50 absorbance reader

    Infinite 200 PRO - Application Notes

    Dynamic interactions between proteins are key mediators of multiple cellular functions and are involved in almost all biological processes. Accordingly, they are valuable targets for the development of novel drug therapies. In this context, bioluminescence resonance energy transfer (BRET), developed by Promega, is a well-established detection technology for the investigation of protein-protein interactions in living cells. However, its application has so far been somewhat hampered by its limited dynamic range and sensitivity.

    ...

    One of the most used methods for determining nucleic acid concentrations is based on measuring the optical density of a sample at 260 nm (OD260). According to the Lambert-Beer law, the amount of absorbed light is proportional to the concentration of the sample, and to the pathlength the light has to pass through when going through the sample.

    ...

    Implementation on Tecan’s Infinite® multimode reader series

    Introduction

    Adenosine diphosphate (ADP) is an important mediator of cellular metabolism that is essential for maintaining energy levels. The Transcreener ADP2 TR-FRET Red Assay is intended for the detection of ADP production by any kinase or ATPase, and has been developed specifically for high throughput screening applications. The assay is based on the competitive binding of ADP to a monoclonal antibody-terbium conjugate, and uses a homogeneous TR-FRET detection mechanism (1). In the absence of free ADP, excitation of terbium results in energy transfer to a far-red tracer and emission at 665 nm. Any ADP produced by the enzyme of interest displaces the tracer from the antibody, disrupting the FRET reaction (see Figure 1).

    ...

     

    BacTiter-Glo™ Microbial Cell Viability Assay measured on the Infinite® 200 PRO multimode microplate reader


    Introduction

    The BacTiter-Glo Microbial Cell Viability Assay is a luminescence-based assay for the determination of the viability of microbial cells, using quantification of ATP as an indicator of metabolically active, viable cells (1). The chemistry relies on the properties of a thermostable luciferase (Ultra-Glo™ Recombinant Luciferase, Figure 1) and a proprietary formulation for extracting ATP from bacteria. The assay protocol comprises the addition of the BacTiter-Glo reagent directly to the sample, and subsequent measurement of luminescence; cell washing, removal of culture medium, and multiple pipetting steps are not required (Figure 2). The luminescent signal generated is proportional to the amount of ATP, and therefore an indicator of the number of viable cells in the sample. The ‘glow-type’ nature of the assay permits signal measurement over a period of approximately 30 minutes, depending on the type of bacteria and growth medium. The BacTiter-Glo Microbial Cell Viability Assay was tested on the Infinite F200 PRO filter-based multimode reader, using the instrument’s highly sensitive luminescence module.

    ...

    Abstract

    We demonstrate the use of a non-activity-based cytotoxicity probe, CellTox™ Green Dye, which can be added at the beginning of an experiment and employed in real time
    for the kinetic determination of cytotoxicity. We used the HP D300 Digital Dispenser, available from Tecan, for noncontact dispensing of test compound and the Tecan Infinite® M200 PRO with Gas Control Module (GCM™) for kinetic measurement of cytotoxicity in HepG2 cells over 72 hours.
     

    Introduction

    In vitro cytotoxicity is largely influenced by test article concentration and the exposure period with cells. The diversity of kinetic response can complicate conventional cytotoxicity endpoint assay determinations because most assay reagents are formulated to measure enzymatic biomarkers that are susceptible to time-dependent decay. Using DNA binding as an indicator of cytotoxicity mitigates this issue, providing a robust method of detection for time course experiments. CellTox™ Green Dye is an asymmetric cyanine
    dye that measures cytotoxicity as a result of compromised cellular membrane integrity (Figure 1). When the dye binds to DNA, green fluorescence is enhanced. The signal intensity is directly correlated to the degree of change in the membrane integrity. The dye can be added at the time of cell plating or compound treatment and the fluorescence monitored kinetically over a period of up to 72 hours. Alternatively, the dye can be added at the conclusion of compound treatment and an endpoint measurement taken.

     

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    Measuring oxygen radical absorbance capacity with the Infinite® 200 PRO multimode reader

    Introduction

    Reactive oxygen species (ROS) are generated as natural byproducts of the cellular metabolism. They are involved in various biological processes, functioning as important signal mediators. However, excess intracellular levels of ROS may result in cell and tissue damage, and are associated with degenerative diseases, most notably cancer. In healthy individuals, intracellular antioxidant systems maintain ROS levels below a critical threshold, permitting essential ROSmediated signaling processes to function, but  preventing ROS overproduction and potential tissue damage [1]. Cells that fail to compensate and neutralize heightened ROS levels die by apoptosis to avoid passing on ROS-caused DNA damage to daughter cells. Any dysfunctions in the cellular antioxidant systems can therefore have serious consequences. In addition to the cells’ own antioxidant systems, various studies have suggested a relationship between an antioxidant-rich diet and a good health status, implicating that the consumption of antioxidant-containing foods can help to maintain health and even prevent certain diseases [2].

    A well-established and reliable method to determine the antioxidant capacity of a  substance is the oxygen radical absorbance capacity (ORAC) assay [3]. It is based on the inhibition of oxyradical-induced oxidation of 2,2’-azobis-(2- methylpropionamidine) dihydrochloride (AAPH) by substances with antioxidant properties. Peroxyl radicals produced in a time-dependent manner during the thermal decomposition of AAPH will quench the fluorescence signal. In the presence of a substance with antioxidant  properties the fluorescence reduction is inhibited, depending on the substance’s ORAC capacity. The dynamics of the signal inhibition, expressed as the area under the curve (AUC), are used to quantify the antioxidant capacity, expressed as the ORAC value, by comparing the sample AUC to an antioxidant standard curve generated with Trolox, a water-soluble vitamin E analog. This application note describes the use of the Infinite® F200 PRO in combination with a commercially available ORAC assay kit, using different beverages as antioxidant samples...

     

     


    Low volume, high sensitivity ELISAs using automation-compatible OptiMax™ plates


    Introduction

    One of the challenges in life sciences research today is to discover methods for running key assays more quickly, more reliably and using lower volumes of reagents and sample, but still with improved sensitivity. One area where this particularly holds true is for traditional enzyme-linked immunosorbent assays (ELISAs), whose application provides a useful measurement of antigens, including cytokines and a host of other biomarkers.

    ELISAs are considered one of the most useful secondary or tertiary type assays in drug discovery, because they elucidate specific cellular pathways and associated mechanisms of action for target genes, proteins or small molecules. They are equally important to clinical biology laboratories, as they enable determination of biomarker concentrations in unknown biological samples.

    There have been a number of attempts to replace the traditional plate-based ELISA with microfluidic-based technology, but in general these have all suffered from the need for specialized liquid handling systems. Until now, microfluidic technology has not been adapted to the SBS plate footprint, and could not make use of the plate-based liquid handling and detection instrumentation found in many life science laboratories.

     

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    Fluorescence-based drug sensitivity testing using 3D tumor microtissues and the Infinite® M200 PRO monochromator-based multimode reader

    Introduction

    Cell viability assays are commonly used in cell biology and drug discovery to characterize cell responses to endogenous and exogenous factors or substances, such as cytotoxic drugs and environmental changes (1). Generally, cell viability is assessed using vital dyes, from which viability can be concluded either directly or indirectly. This method, although often used, is labor intensive and tedious. Automated alternatives include electric cell counters (1) and flow cytometers which, although accurate, are associated with sophisticated equipment and high assay costs, and require technical expertise. As a result, fluorescence-based cellular assays are becoming increasingly popular, due to their sensitivity and versatility.

    The Infinite M200 PRO offers enhanced fluorescence intensity reading for cell-based and biochemical applications, with a range of features designed to improve sensitivity and inter- /intra-well reproducibility. Functions such as orbital shaking, temperature control and enhanced fluorescence bottom reading with the optimal reading (OR) function ensure excellent performance and reliability.

    To further improve the predictive power of in vitro cell-based assays, cell models have to mimic more closely the three dimensional (3D) structure of organs and tissues in vivo (2). Scaffold-based 3D cell culture approaches often suffer high background fluorescence, due to autofluorescence from the scaffold biomaterials. Scaffold-free microtissues are therefore ideally suited to this application, offering tissue-like structures while allowing researchers to take advantage of embedded fluorescent reporter technology.

    Combining Tecan’s Infinite M200 PRO with InSphero’s organotypic microtissue tumor model (which harbors fluorescent reporter proteins) provides a scalable system to assess drug sensitivity in complex 3D cell culture models. This allows long-term, tissue-based analyses – such as cell proliferation studies – offering reproducible measurements over time....

    Walkaway growth monitoring of Helicobacter pylori in the Infinite® 200 PRO reader with Gas Control Module (GCM™)

     

    Introduction
    Growth analysis of microorganisms via OD absorbance measurements at 600 nm is key to many different research areas. In the past, this has been a time-consuming and
    labor-intensive procedure. Tecan’s Infinite 200 PRO multimode reader can now provide all essential growth conditions – including continuous shaking, temperature control, O2 and CO2 regulation and humidity stabilization – within the measurement chamber, eliminating the need to transfer microplates between the cell incubator and the microplate reader (1). This enables simultaneous incubation and signal detection without the need for any manual intervention, even for microorganisms which need very specific environmental conditions, such as facultative anaerobic bacteria.

    In this collaborative study, Tecan and its partners have analyzed the growth of the human pathogen and class-I carcinogen Helicobacter pylori over a period of 28 hours.

    As a microaerophilic organism, H. pylori needs low atmospheric oxygen concentrations for optimized growth, and normally colonizes human mucosa, where it can cause certain
    types of stomach disorders and cancer. To mimic these physiological conditions, it is a prerequisite for the incubation/detection device to have the capability to control atmospheric O2 levels.

    Bacteria were incubated at 37 °C inside the measurement chamber of the Infinite 200 PRO reader, with continuous shaking, 10 % CO2 and varying O2 levels (normoxic  (control), 5 % and 10 % O2). The proliferation was monitored by measuring sample absorbance at 600 nm, and the fluorescence of GFP-transformed H. pylori. To minimize
    evaporation effects during the long incubation period inside the reader, Tecan  recommends using the Nunc Edge 96-well plate, which has been shown to minimize evaporation when used in combination the Infinite 200 PRO and the GCM (2, 3)...

     

     

    Detection of tyrosine kinase activity using the Infinite F200 PRO’s new AlphaScreen function

     

    Introduction
    AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay) is a bead-based screening technology developed for fast, reliable and cost-effective detection of biomolecular interactions. It utilizes the energy transfer between donor and
    acceptor beads that occurs when these are brought into close proximity due to a binding event between their coupling partners. As a result, a strong luminescent signal is generated that can be detected in a wavelength range of 520-620 nm [1].

    Tyrosine kinases are important mediators of cellular processes such as signal  transduction, cell growth and apoptosis. They have been reported to be involved in a
    number of diseases associated with excessive cell proliferation – including  atherosclerosis and cancer – and are therefore often targeted in drug development and highthroughput screening (HTS) approaches.

    AlphaScreen-based phosphotyrosine assay kits, for example P-Tyr-100, have been developed for reliable and sensitive detection of kinase activity. The AlphaScreen signal is dependent on the extent of tyrosine kinase phosphorylation.[2].

    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) techniques such as HTRF®. In addition, the Infinite F200 PRO is now capable of measuring AlphaScreen- and AlphaLISA®-based assays...

     

     

    Detection of human immunoglobulin G (IgG) using the Infinite F200 PRO’s new AlphaLISA function

     

    Introduction

    AlphaLISA is a homogeneous, no-wash alternative to conventional ELISAs based on PerkinElmer’s bead-based Alpha (Amplified Luminescent Proximity Homogeneous
    Assay) technology. AlphaLISA assays can be set up as sandwich or competitive immunoassays to detect and quantify molecules of interest in biological samples [1].


    High energy excitation of photosensitizer molecules within the AlphaLISA donor beads at 680 nm converts ambient oxygen to singlet oxygen, which in turn is able react with the chemistry in the acceptor beads if these are in close proximity. A cascade of energy transfer steps ultimately results in the generation of a strong luminescence signal at 615 nm, indicating specific binding between the molecules attached to the two bead types. The fluorophores embedded in the AlphaLISA acceptor beads produce a narrower bandwidth signal than the acceptor beads used for classical AlphaScreen® assays.

    This makes AlphaLISA assays less prone to signal interference at wavelengths of <600 nm, increasing the sensitivity and robustness of the assay. The use of dedicated AlphaLISA optics permits the analysis of target molecules in blood and serum by  drastically reducing the effect of hemoglobin within a sample.

    The new module for AlphaScreen and AlphaLISA assays is the latest addition to the Infinite F200 PRO’s multimode functionality. In addition to established reading modes,
    including absorbance, fluorescence top/bottom, single and dual luminescence, fluorescence polarization (FP) and timeresolved fluorescence resonance energy transfer (TR-FRET) techniques such as HTRF® and LanthaScreen™, the Infinite F200 PRO now offers a module for Alpha-based assays, tailored to the needs of low to medium throughput applications and basic research...

    Fluorescence-based drug sensitivity testing using 3D tumor microtissues and the Infinite® M200 PRO monochromator-based multimode reader

    Optimizing bacterial growth studies on the Infinite® 200 PRO multimode reader platform using permanent shaking and heating

     

    Long-term cell-based kinetics using Tecan’s GCM™ and
    the Thermo Scientific Nunc Edge plate

    Enabling long-term cell-based assays with eukaryotic cells via controlled CO2 partial pressure inside the reader

    Cell-based applications are central to life science research. They may range from cytotoxicity, proliferation, apoptosis and GPCR signaling assays to high-throughput screening (HTS) drug discovery applications. Adherent cell types are typically analyzed through the well bottom in order to bring the cell monolayer as close as possible to the detector and avoid unspecific fluorescence noise by the overlying growth medium. For this reason it is essential to guarantee illumination and reading of the entire well bottom, since cells are not always homogeneously distributed over the growth surface.

    In vivo kinetic studies of drug uptake across the gastro-intestinal tract (GIT) and blood-brain barrier (BBB) are valuable tools for assessing bioavailability to prospective targets. These are relatively expensive and time consuming assays which are conducted sparingly. pION Inc. has introduced a parallel artificial membrane permeability assay (PAMPA) which has recently gained popularity as a novel, cost-effective high-throughput assay capable of rapidly screening compounds for their permeability characteristics in early drug discovery. In this note we describe the easy implementation of Tecan´s Infinite M200 with its absorbance scanning feature for PAMPA sample analysis.

    This application note describes the successful implementation of the fluorescence intensity based Technothrombin® TGA assay on Tecan´s Infinite M200 monochromator based multimode detection system.
    The amount of generated thrombin is dependent on the number of micro particles present in the sample. PFP showed a delayed and lower Thrombin formation compared to PPP.

    In this application note we describe the use of Tecan’s HydroFlexTM washer equipped with a magnetic bead plate carrier for fast purification of a large number of samples using magnetic beads.For the detection of the luminescence signal Tecan’s Infinite® F200 multimode reader was used.

    Here we describe the preparation and evaluation of RNA samples from a novel population of mouse keratinocyte stem cells marked by Lgr5+ expression. We used tools specifically designed for handling and measuring low RNA quantities including RNA purification and the subsequent quantification with the Infinite® M200 NanoQuant.

    This note describes the implementation of the Infinite® 200 multimode reader and the associated NanoQuant Plate™ for fluorescence-based DNA quantification in small volume samples using Pico Green®, an ultra-sensitive fluorescent dye for the quantitation of double-stranded DNA.

    Among the devices suitable for measurements of small-volume samples Tecan’s Infinite® 200 NanoQuant in combination with the NanoQuant Plate™ offers uncompromised performance for absorbance-based nucleic acid quantification and assessment of labeling efficiency and, in addition, can be upgraded at individual convenience with fluorescence and luminescence reading functions.The present note describes the implementation of the NanoQuant Plate™ for protein measurements with regard to essential assay parameters such as linearity, uniformity, and reproducibility.

    With its NanoQuant Plate™, Tecan provides a new tool for reproducible and sensitive quantification as well as purity check of nucleic acids.
    For quality control of oligonucleotide labeling reactions in real-time PCR assays and in array hybridization experiments using dye-labeled probes, the labeling efficiency is an impor-tant parameter to evaluate results. The NanoQuant Plate can be easily used to determine the labeling efficiency of nucleic acid probes.

    Protein quantification is often required before proceeding with protein samples for isolation, chromatographic or electro-phoretic analysis, or immunohistochemical methods. Two different techniques are generally used for colorimetric detection and quantification of proteins: protein-dye binding and protein-copper chelation. In this note we describe the use of Tecan’s Infinite® F200 and Infinite M200 instruments for easy and sensitive protein quantification using different absorbance-based assays.

    For protein assays in general, and especially for the modified Lowry protein assay, the reader-injector system is an ideal solution avoiding pipetting and workflow errors, and resulting in consistent and traceable data.Tecan´s Infinite® 200 instrument series extended with the injector system offers a multi-functional system, from injection of reagents, to mixing, incubation and measuring, with all steps performed within one instrument set-up.

    Infinite M1000 PRO - Application Notes

    Introduction

    The SciFlow™ Multiwell Cascading Fluidics Cell Culture System is a versatile system that enables the application of fluidic motion and gradient toxicant exposure to cell-based assays in a proprietary microplate reader-compatible 96-well format. Time-resolved dynamic exposure scenarios afforded by the SciFlow System is more in vivo-like and could enable more accurate assessment of adaptive vs toxic mechanisms. Moreover, the dynamic exposure scenario allows better prediction of concentration thresholds for response rather than extrapolating from endpoint dose-response curves.

    The SciFlow System is an SBS formatted microtiter plate designed to be compatible with plate-readers, imagers, and automated liquid handling systems. In order to enable efficient flow of fluids across the plate, the SciFlow System was designed with variable well-heights. The wells are elevated at one end of the plate and subsequently the bottom surface of each well is 0.5mm lower than the previous well. This allows for a cascading flow of fluids across the plate (Figure 1). The Tecan Infinite® M1000 Pro plate reader features the ability to empirically determine the optimal height of each measurement.

    ...

     

    NanoQuant Plate™ for use on Infinite® M1000 and Infinite F500 multimode microplate readers

    Many current genetic and forensic investigations, for example microarray-based hybridization experiments, require the detection and quantification of very small amounts of nucleic acids. Microarray-based hybridization experiments use dye-labeled DNA or RNA probes, requiring concentration and labeling efficiency measurements to determine the degree of dye incorporation.

    This application note describes the successful validation and implementation of the PredictorTM hERG Fluorescence Polarization Assay for research purposes within drug discovery on the Tecan Infinite® M1000 premium Quad4 Monochromators™ based multimode detection system. Tecan’s Infinite M1000 offers an easy-to-use and flexible way of accessing fluorescence polarization data.

    LS Reloaded - Application Notes

    The gain in the LS Scanner is a direct representation of the voltage of the PMT. It can be set to any value between 70 and 255. Because there is a linear relationship between gain and PMT voltage, this corresponds to PMT voltages between 330 and 1200 Volt.

    In the following we describe MWG's processes and how we tackled the challenges outlined above. In a high throughput production and service facility running in an ISO 9001 environment this only can be achieved with a high degree of automation. With the automated Tecan LS 200 Laser Scanner and the Tecan HS 4800™ Hybridization Station MWG found suitable tools to ensure the required throughput and at the same time gain maximum control and reproducibility required to generate high quality data.

    Spark - Application Notes

    Temperature is a major factor affecting the rate and dynamics of biochemical reactions. The Spark® multimode reader’s ingenious Te-Cool™ module permits measurements even below ambient temperature, thereby limiting the effects of temperature increases in the laboratory on the dynamics of assays. It helps to stabilize temperature-dependent assays and makes the results more reproducible and reliable.

    The Optimal Read function for fluorescence bottom measurements is designed to enable uniform well illumination during excitation while and whole-well emission for 6-to 384-well plates. Performing multiple measurements on spatially separated spots arrayed across each well ensures that even heterogeneous cell layers can be effectively and reproducibly analyzed. This application note shows that the Optimal Read function produces the most reliable results for the analysis of adherent cell types, giving the highest well-to-well uniformity and best sensitivity for cell-based applications.

    ...

    Homogeneous Time-Resolved Fluorescence (HTRF) is a time-resolved fluorescence resonance energy transfer (TR-FRET) assay system developed by Cisbio Bioassays, and offers a reliable technique for high throughput screening (HTS) and the analysis of molecular interactions. The assay is based on energy transfer between a long-lived europium or terbium cryptate donor and a variety of red or green acceptors, and combines the advantages of FRET and time-resolved fluorescence (TRF).

    For many cell biology applications – such as the identification and separation of high expression clones or knock-down candidates, and small-scale antibody or protein production – it is important to ensure that cell lines originates from a single cell, i.e. they are monoclonal. This can be a major bottleneck, especially for academic laboratories, as dedicated automated microscopy systems are both very expensive and complex to operate, meaning verification of monoclonality is often performed by manual microscopy.

    Tecan’s Spark multimode microplate reader was used for the real-time analysis of human stem cell proliferation and differentiation, using established assays for cell viability, proliferation and apoptosis assessment in a reliable and convenient manner.

    Tecan’s Spark multimode microplate reader was used to identify metabolic changes during the differentiation of human mesenchymal stem cells and to assess and quantify cell viability, cytotoxicity and apoptosis, using several well-established and reliable cell-based assays developed by PromoCell.

    Cell migration is a central process in the development and maintenance of multicellular organisms, with tissue formation during embryonic development, wound healing and immune responses all requiring the orchestrated movement and assembly of cells.

    Assaying cells in an appropriate window of time after a compound-­mediated cytotoxic event can be extremely challenging. This note describes the automation for kinetic monitoring of cytotoxicity and viability using three Promega assays for viability, cytotoxicity and apoptosis assessment with the Spark multimode reader.

    Keep control of crucial experimental conditions with the first air conditioning system for a multimode reader – enabling stable measurement chamber temperature down to 18°C.

    Tecan’s Spark multimode microplate reader with integrated cell imaging simplifies cell culture, featuring quality checks and signal normalization to cel lconfluence, thereby boosting automation of cell-based studies.

    This application note describes the implementation of the AlphaLISA technology on the Spark multimode reader and its use for the detection of human immunoglobulin G (IgG) using the AlphaLISA IgG Assay Kit.

    This application note describes the implementation of the AlphaScreen technology on the Spark multimode reader and the detection of tyrosine kinase activity using the AlphaScreen P-Tyr-100 Assay Kit.

    This application note describes the successful implementation of AlphaScreen on the Spark multimode reader, as well as the performance of the instrument with regards to measurement sensitivity, uniformity and typical read times.

    HTRF is a time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay system from Cisbio Bioassays, and is becoming increasingly popular for the analysis of various molecular interactions and binding studies. HTRF assays place high sensitivity demands on multimode readers. The Spark offers a number of features to meet these demands and to ensure optimal assay performance, including Tecan’s unique Fusion Optics which combine the sensitivity of filters and the flexibility of monochromators (MCRs) in one instrument. In addition to entirely filter- or MCR-based measurements, a combination of the two can be used simultaneously within one measurement.

    Cell counting, viability analysis and determination of size distribution are routinely performed by researchers across multiple disciplines, from standard cell passaging for downstream experiments to using insect cells infected with baculovirus to produce recombinant proteins, or determining the differentiation capacity of stem cells. Tecan’s Spark multimode reader has an integrated, bright field cell imaging module. This patent-pending system enables label-free cell counting, size distribution determination and trypan blue-based cell viability analysis with an easy-to-use, disposable Cell Chip™. In this study, the analysis of cell concentration, cell viability and size distribution for the determination of the optimal protein production rate in recombinant Sf21 cell cultures is presented.

    Spark Stacker

    Sunrise - Application Notes

    In this application note we describe the use of TECAN Magellan™ software together with a TECAN SUNRISE™ microplate reader for spectrophotometric measurements of an enzymatic assay of alkaline phosphatase in glycine buffer. From the kinetic raw data the kinetic parameters: maximum reaction velocity Vmax and the Michaelis constant Km are calculated.

    This application note presents a new PCR and colorimetric detection approach for easy and reliable detection of mutations using Tecan´s 96 micro plate Sunrise™ absorbance reader. For high-throughput analysis of genomic mutations or polymorphism screenings, the MutectorTM STA primer extension assay in combination with the Sunrise reader is an ideal combination.

    ULTRA Evolution - Application Notes

    Detection of the Mitochondrial Potential Sensor JC-1
    Tecan Ultra Evolution, Safire and GENios Pro


    JC-1 (Figure 1) is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~ 525 nm) to red (~ 590 nm). Mitochondria depolarization is indicated by a decrease in the red to green fluorescence intensity ratio. The potential-sensitive color shift is due to concentration-dependent formation of red fluorescent J-aggregates. JC-1 can be used as an indicator of mitochondrial potential in a variety of cell types as well as in intact tissues and isolated mitochondria.

    The ratio of green to red fluorescence is dependent only on the membrane potential and not on other factors such as mitochondrial size, shape and density that may influence single-component fluorescence signals. The fluorescence ratio detection allows comparative measurements of membrane potential to be made and...

    Detection of Calcein AM and Hoechst 33342
    Tecan Ultra Evolution, Safire and GENios Pro


    Calcein AM (acetoxymethyl ester of Calcein) is one of the premier indicators of cell viability due to its superior cell retention and the relative insensitivity of its fluorescence to physiological pH values. Live cells may be distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant Calcein AM to the intensely fluorescent Calcein. Calcein, which is the hydrolysis product of Calcein AM, is a polyanionic fluorescein derivative. Calcein is well retained within live cells, producing intense uniform green fluorescence in live cells (Molecular Probes: C-1430). Hoechst 33342, a bisbenzimide dye, is a cell membrane permeant, minor groove-binding DNA stain that...

    Detection of the Photosensitizer Hypericin
    Tecan Ultra Evolution, Safire and GENios Pro


    Hypericin is a powerful, naturally occurring photosensitizer that is found in Hypericum perforatum plants, commonly known as St. John's wort. Hypericin (HY) is a polycyclic phenanthroperylenedione, which in cells binds mostly to the cellular membrane and can be metabolized rapidly in vivo with no toxic properties.

    In recent years, there has been an increased interest in Hypericin as a potential clinical anti-cancer agent, since several studies established its powerful in vivo and in vitro anti-neoplastic activity when irradiated. Investigations of the molecular mechanisms underlying Hypericin photocytotoxicity in cancer cells have revealed that this photosensitizer can induce both apoptosis and necrosis in a concentration and...

    Detection of Green Fluorescent Protein (eGFP)
    Tecan Ultra Evolution, Safire and GENios Pro


    spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequorea victoria, or from the sea pansy, Renilla reniformis. Its role is to transduce the blue chemiluminescence of aequorin, into green fluorescent light by energy transfer. The molecular cloning of GFP cDNA and the expression of GFP as a functional transgene has opened exciting new avenues of investigation in cell, developmental and molecular biology. GFP can function as a protein tag, as it tolerates N- and C-terminal fusion to a broad variety of proteins many of which have been shown to retain native function. Highly specific intracellular localization including the nucleus, mitochondria, secretory pathway, plasma membrane and cytoskeleton can be achieved via fusion both to whole proteins and...

    Chroma-Glo® Assay on Tecan ULTRA Evolution and GENios Pro
    Dual Reporter Gene Luciferase Assay System, Promega


    This technical note introduces the ULTRA Evolution and the GENios Pro, both multifunctional plates readers of Tecan, for dual color luminescence measurement. The Chroma-Luc® Technology, a homogenous dual reporter gene assay from Promega (US), was successfully evaluated using lysates containing the Chroma-Luc® luciferases as demo systems.

    The Chroma-Luc® Technology, which consists of the Chroma-Luc® Reporter Vectors and the Chroma-Glo® Luciferase Assay System, is a homogenous dual-reporter gene assay. Such kind of genetic reporter systems are widely used in cell biology research and pharmaceutical discovery in order to test a variety of experimental conditions or a large number of chemical compounds for...

    A New Generic Luminescent Protein Kinase Assay
    Suitable For High Throughput Screening


    Protein Kinases and their ability to phosphorylate proteins play key roles in the signal transduction pathways of many diseases such as cancer, arthritis and diabetes1. Thei importance of protein kinases makes them common targets for many High Throughput Screening departments (HTS) within the pharmaceutical industry. Current screening technologies such as HTRF and SPA employ the use of phospho-state specific antibodies or radioactive beads. The need for new antibodies and beads for each kinase/substrate pair makes these assays expensive and time consuming to develop and run. Cambrex have developed PKLight(™), a non-radioactive, homogeneous, robust and simple assay suitable for the screening of potentially all protein kinases in 96, 384 and 1536-well formats. This technology utilises Luciferase bioluminescence to measure ATP consumption as a result of kinase phosphorylation of the target substrate. The assay can be easily optimised for...

    Implementation of HTRF® on Tecan Ultra Evolution
    Human TNFα and cAMP kit, CIS bio international


    The current technical note introduces two applications, implemented and validated on the ULTRA Evolution, Tecan’s multifunctional microplate reader. These applications utilise HTRF® (Homogeneous Time-Resolved Fluorescence) technique to generate the assay readout (CIS bio international, France).

    HTRF® (Homogeneous Time-Resolved Fluorescence) technology is based on the energy transfer between two fluorescent labels, a long-lifetime Eu3+-cryptate donor and the XL665 acceptor (chemically modified allophycocyanin) (1). This technique combines both, time-gated fluorescence (commonly referred to as time-resolved fluorescence) and...

    Fluorescence Lifetime (FLT)
    A comparative report which demonstrates FLT efficacy in HTS
    Relative to other detection/assay technologies


    throughput screening (HTS), with the emphasis now shifted from numbers of compounds screened to the quality of the data and higher information content. This shift puts greater reliance on the assay or detection technology and its robustness in the screening environment.
    Assay robustness is the term applied to how an assay signal tolerates interferences in a real screening campaign. Critical to this assessment are the performance parameters (Z’, signal window and reproducibility)1 by which assay quality of the control signals from an HTS are judged. But of equal importance, is the ability of the assay signal to avoid perturbation by the numerous non-specific effects that...

    Fluorescence Lifetime (FLT) Measurements
    An introduction to the technique


    This technical note introduces the basic principles of the new technique Fluorescence Lifetime (FLT). It will provide a brief overview of both the theoretical and the experimental requirements, followed by an application-focussed discussion of the data interpretation process.

    1. The role of microplate readers in HTS
    Common microplate readers allow the user to monitor biological or biochemical assays which have a marker or label incorporated which interacts with light. Among the frequently used readout modes are absorbance, fluorescence intensity (FI), fluorescence polarisation (FP), time-gated fluorescence (TRF) signals. None of these methods can be applied to all possible assay situations, which is why a complementary palette of methods is necessary to cover as...

    DNA Oligonucleotide Hybridisation
    Automatic Detection of Fluorescence Lifetime with ULTRA Evolution


    Hybridisation of two complementary DNA single-strands into one double strand is a fundamental biochemical reaction. The extent of this reaction can be inhibited or modulated by modifications of one of the binding partners (e.g. single nucleotide polymorphism) or by adding extraneous chemicals or proteins.
    Within this technical note we utilise the well understood hybridisation reaction as a model system to demonstrate how to use Fluorescence Lifetime as a signal for monitoring (bio-)chemical reactions. Elsewhere we laid out the theoretical and technical basis for lifetime experiments (ref 1). In short, the light emitted from fluorescent labels is characterised by several parameters, among which one is the Fluorescence Lifetime. This parameter is highly susceptible to changes...

    Drug Screening Using Fluorescence Lifetime Analysis
    Human α-Thrombin Aptamer


    Tecan has recently introduced a fully automated Fluorescence Lifetime Analysis (FLT) platform for detection in microplates. Several preceding technical notes have detailed the methodical background of this emerging technique [1, 2, 3]. In brief, fluorescence lifetime can be utilized to report directly about the status of biochemical reactions, like receptor-ligand binding or enzymatic activity. FLT provides an inherently more robust signal when compared to standard spectroscopic readouts like absorbance or fluorescence intensity. It is therefore regarded to be especially suited to drug-compound screening campaigns.
    In this note, we apply FLT to another relatively new area, an aptamer based antagonist screen for human α-thrombin. Aptamers are single-stranded oligonucleotides that,...