Fast multiple plate replication

Use of 384-channel parallel dispensing

F. Hoffmann-La Roche AG (Roche), Switzerland recently added an innovative system called nanoAPR™ (Automated Plate Replicator), a high-throughput low-volume plate replication system, to its core Compound Management operations in Basel. The nanoAPR is the result of a joint project between REMP AG (REMP), Tecan Schweiz AG (Tecan) and Roche to pro-vide a solution for the production of large num-bers of replicas of Roche’s HTS libraries. The nanoAPR has been used in the first quarter of 2005 to successfully generate approximately 45’000 replicas of 384-well plates. This note describes the challenges encountered during this task and how they have been ad-dressed. It demonstrates the dilution of...

Running Alvetex® Scaffold in 96-well plate format on the Freedom EVO®

Introduction

Experiments performed in a classical two-dimensional (2D) cell culture system have resulted in a large body of knowledge about basic life science. This type of research began almost 130 years ago, and has provided numerous answers about intrinsic cellular biology and developmental molecular processes, as well as enabling breakthroughs in regenerative medicine and insights into diseases and drug discovery. However, the 2D cell culture systems used so far have several drawbacks: the morphology, proliferation, metabolism and expression profiles of cells grown in 2D systems are very different to cells in living tissues.

br>Technological advances in the last couple of years have enabled 3D scaffolds to be engineered that offer a real proxy for the in vivo environment which can be easily and routinely used in a cell culture laboratory. The Alvetex Scaffold (Figure 1) is a 200 μm thick, highly porous and uniform polystyrene scaffold, designed and developed by Reinnervate (Sedgefield, UK).

Alvetex Scaffold enables cells to develop natural, in vivo-like intercellular interactions, providing an ideal environment for real three-dimensional growth and nutrient exchange that is comparable to intra-capillary exchange in living tissues...

Freedom EVO® platform with Pickolo™ colony-picker for biofuel production
 

Introduction
Abengoa Bioenergy New Technologies, based in Seville, Spain, is an international company applying innovative solutions for biofuel production. In this context, the Company was seeking to establish and automate a screening process to determine cellulase activity in mutant strains of cellulaseproducing fungi.

Since manual screening processes are very costly and time consuming, automation was a prerequisite for a project of this magnitude. Abengoa established close collaborations with Biomar Microbial Technologies and Mejoran Lab Automation to create the optimal solution. Biomar, located in León, Spain, is a pioneer in research into micro¬biological applications to generate sustainable solutions in sectors such as healthcare, agriculture and renewable energy, and therefore has a solid background in generation, screening and bioactivity profiling of microbial collections. Mejoran, based in Madrid, Spain, is a laboratory automation solution provider.

The first step in the three-stage screening process was selection of colonies of different fungal mutants and their transfer to a 96-well plate to create microinocula for  further fermentation. The second step involved fermentation of the different mutants in a 96-well plate format. Finally, the cellulase-producing capability of the different mutants  was evaluated.
 

A Freedom EVO 150 workstation with SciRobotics’ Pickolo colony-picker module proved the ideal solution for performing both the fungal selection and the subsequent screening of different culture conditions. The Freedom EVO workstation can easily identify, select and pick fungal colonies based on user-selected criteria. Additionally, Freedom  EVOware® and the Pickolo software allowed Mejoran to define a unique picking procedure that achieved 100 % microinoculum growth for subsequent experiments. This was accomplished by picking from the perimeter of the fungal colonies, rather than from the center, in a sweeping motion which optimized the amount of biomass taken up by the tip.

...

Processing time is about 1.5 h for 24 samples from up to 400 mg of plant starting material. The A260/280 ratio as a typical indicator of nucleic acid purity is generally in the range of 1.9 and typical yields are approximately 80 μg per 200 mg of starting material from fresh wheat leaves. Hence, full automation of the nucleic acid extraction procedure on a Tecan Freedom EVO workstation streamlines laboratories’ workflows and allows for reliable and fast extraction of high quality plant genomic DNA.

...

Rapid extraction of genomic DNA from a variety of food and feed samples with NucleoSpin® 8/96 Food columns and the Freedom EVO®

Vet Food Agro Diagnostics Sdn Bhd (VFAD) is a service laboratory based in Malaysia, which provides a wide range of screening and testing services to the veterinary, food, agriculture and agro-based industries across Southeast Asia. VFAD works very closely with local government authorities, such as the Malaysian Department of Veterinary Services (DVS), on the control and prevention of infectious animal diseases, as well as a variety of food safety programs. This work has led to numerous awards and recognitions, as well as development of a number of patent-pending inventions.

The Landeslabor Berlin-Brandenburg (LLBB, State Laboratory of Berlin-Brandenburg) in Frankfurt/Oder, Germany, is an investigative institution dedicated to the protection of human and animal health, as well as of the environment and natural resources. The LLBB has been established in January 2009 to combine the laboratory resources of the states of Berlin and Brandenburg.

First described in the US in 1987, Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral swine disease that causes a range of reproductive and respiratory disorders. The major component of the PRRS is reproductive failure, causing premature births, late-term abortions and stillbirths, weak piglets, decreased farrowing rates and delayed return to estrus, with the consequence of significantly reduced production rates. Respiratory problems often lead to secondary infections, and cause increased mortality in young piglets.

Avian influenza is a bird disease caused by the highly pathogenic influenza virus. Influenza viruses are inherently unstable and, as they lack a genetic proof-reading mechanism, small errors that occur when the virus copies itself go undetected and uncorrected. Specific mutations and evolution in influenza viruses cannot be predicted, making it difficult if not impossible to know if or when a virus such as H5N1 might acquire the properties needed to spread easily and sustainably among humans. This difficulty is increased by the present lack of understanding which specific mutations would lead to increased transmissibility of the virus among humans (www.who.int). Here we describe an automated method for nucleic acid extraction and avian influenza diagnosis.

Your easiest way to optimal results!

Tecan’s multimode readers are symbolized by a hummingbird, the embodiment of speed, precision, and color. Like the plumage of the hummingbird, our monochromators cover the entire spectrum of the visible light. Hummingbirds can hover in mid-air to target flowers even smaller than a well of a 96-well plate, mirroring the precision of instrument measurements. The hummingbird’s heart rate, which can reach as high as 1,260 beats per minute to support the rapid beating of its wings of up to 80 times per second, represents the exceptional speed and throughput rate of Tecan’s multimode readers.

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Reliable extraction of buffy coat from human blood on a Freedom EVO® platform

Introduction

The recent advances in personalized diagnostics continue to inspire a wealth of personalized treatments, resulting in state-of-the-art personalized heathcare. The Integrated Biobank of Luxembourg (IBBL), a newly founded independent, not-for-profit biobank, designed for a new era of research and the next generation of healthcare, is at the forefront of these medical advances. Research into personalized medicine requires high quality samples (including tissue and body fluids, such as blood, blood fractions and saliva) and clinical data from large numbers of patients, collected by biobanks, to translate today’s discoveries into future personalized medical care innovations. As a world class EU biobank, the IBBL provides a wide variety of the highest quality samples, alongside cutting-edge technology, to accelerate clinical research and improve public health.

A valuable body fluid for diagnostic purposes is the buffy coat, a layer of leukocytes and platelets that forms between the erythrocyte layer and the plasma when unclotted blood is centrifuged or allowed to stand. Typically, the isolation of the buffy coat from whole blood has been a tedious and lengthy manual process. Researchers at the Integrated Biobank of Luxembourg have tested and evaluated the feasibility and efficiency of extracting the buffy coat using an automated process on Tecan’s Freedom EVO platform.

The automation of buffy coat extraction is challenging. In addition to the blood volumes in the individual blood collection tubes varying, important inter-individual variations in the quantity, viscosity and texture of the buffy coat layer itself, for example due to leukocyte increase upon infection, can be observed. Tecan’s Freedom EVO platform, equipped with a Tube Inspection Unit (TIU), has been optimized for automated buffy coat extraction, giving superior results compared to the manual process...

Automated culturing and differentiation of pluripotent stem cells into hematopoietic cells

Introduction
Many different research fields – including investigation of early human development processes, disease research, drug discovery and cellular therapy applications – require appropriate in vitro models which can accurately mimic in vivo processes to provide biologically relevant results. Pluripotent stem cells (PSCs) offer a powerful system for creating in vitro models to address these questions. PSCs can be proliferated indefinitely in an undifferentiated state and, when induced with appropriate growth factors and cytokines, can mature into virtually any of the cell types found in the human body.

Currently, differentiation of either human embryonic stem cells (hESCs) or induced PSCs (iPSCs) relies on the use of serum or co-culturing on feeder cell lines. Manual maintenance and differentiation of hESCs or iPSCs is technically challenging, labor-intensive and subject to inherent process variability, and is therefore not a viable long-term solution for scaling up the production of stem cells.

Cellular Dynamics International (CDI), based in Wisconsin, USA, has developed an automated method to produce industrial quantities of stem cells, and their derivatives, to address the needs of the market. This study describes a highly efficient, serum-free, feeder-free automated system for production of iPSCs, and their differentiation into hematopoietic precursor cells (HPCs).

Materials and Methods
The automated system, a Cellerity™500, is based on a Freedom EVO® 200 liquid handling platform, and includes an STX 500 automated incubator with a capacity for 500 plates (LiCONiC); a media storage fridge; an AutoLoader™ for loading flasks; a Cedex™ cell counter (Innovatis); spinner flasks for expansion and seeding of suspension cells; a Robotic Manipulator (RoMa) Arm to handle plates; Multichannel Arm (MCA) and an eight-channel Liquid Handling (LiHa) Arm using fixed tips...

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

Introduction

Establishing protocols for automated harvesting of various cell lines from multiwell plates

Freedom EVO® platform with Pickolo™ colony-picker for biofuel production
 

Introduction
Abengoa Bioenergy New Technologies, based in Seville, Spain, is an international company applying innovative solutions for biofuel production. In this context, the Company was seeking to establish and automate a screening process to determine cellulase activity in mutant strains of cellulaseproducing fungi.

Since manual screening processes are very costly and time consuming, automation was a prerequisite for a project of this magnitude. Abengoa established close collaborations with Biomar Microbial Technologies and Mejoran Lab Automation to create the optimal solution. Biomar, located in León, Spain, is a pioneer in research into micro¬biological applications to generate sustainable solutions in sectors such as healthcare, agriculture and renewable energy, and therefore has a solid background in generation, screening and bioactivity profiling of microbial collections. Mejoran, based in Madrid, Spain, is a laboratory automation solution provider.

The first step in the three-stage screening process was selection of colonies of different fungal mutants and their transfer to a 96-well plate to create microinocula for  further fermentation. The second step involved fermentation of the different mutants in a 96-well plate format. Finally, the cellulase-producing capability of the different mutants  was evaluated.
 

A Freedom EVO 150 workstation with SciRobotics’ Pickolo colony-picker module proved the ideal solution for performing both the fungal selection and the subsequent screening of different culture conditions. The Freedom EVO workstation can easily identify, select and pick fungal colonies based on user-selected criteria. Additionally, Freedom  EVOware® and the Pickolo software allowed Mejoran to define a unique picking procedure that achieved 100 % microinoculum growth for subsequent experiments. This was accomplished by picking from the perimeter of the fungal colonies, rather than from the center, in a sweeping motion which optimized the amount of biomass taken up by the tip.

...

PRE-ANALYTICAL PROCESS AUTOMATION:

AN INTEGRATED WORK CELL APPROACH

First Look at Tecan’s Genesis FE500 Pre-analytical Processor

Analysis was the first aspect of clinical lab work to be automated, not because it was the most labor intensive, but because it provided the greatest overall cost reductions. Pre-analytical processing, representing about 65% of average total manpower allocation, is actually the most labor-intensive aspect of clinical lab work. However, up until now small- to medium-sized labs could not afford the cost or space required to piece together the requisite segments of preanalytical process automation. Now a fully automated, integrated work cell, combining all the essential pieces within a single, small footprint, is available that offers an affordable alternative.

In this article, Professors Robin A. Felder of the University of Virginia’s Medical Automation Research Center and Laurence M. Demers of Pennsylvania State University’s University Hospital Laboratory review their labs' beta test experiences with the Tecan Genesis FE500 pre-analytical processor—a compact, stand-alone system that can automate pre-analytical processing of blood specimens.

Freedom EVO | PCR

Flexible solutions for high-throughput PCR

The Tecan Freedom EVO PCR workstation is a fully scalable and modular system enabling PCR and cycle sequencing reaction preparation as well as normalization applications.The pipetting platform offers unsurpassed flexibility to support virtually all the reaction preparation needs of laboratories whatever the throughput. Reactions may be prepared in micro tubes, 96- or 384-well plate formats. Precise and reliable pipetting of low volumes is performed by the advanced fluidic system including high-performance low volume tips. Contamination-free pipetting of template DNA, DNA primers, and other components (buffers, enzyme, nucleotides) is guaranteed even for the most sensitive amplification procedures by the 4- or 8-tip liquid handling arm or the 96- and 384-multi-channel pipetting option. Extended automation of amplification procedures can be easily achieved by integration of Tecan’s GENios plate reader for nucleic acid quantification and normalization as well as by...

Preparation of viral RNA for veterinary diagnostics

A versatile platform for automating sample preparation for testing virus-based diseases

Laboratory workers at the Chemical and Veterinary Institute (Chemisches und Veterinäruntersuchungsamt Rhein-Ruhr-Wupper, CVUA-RRW) in Krefeld, Germany, provide a number of veterinary diagnostic tests on animal samples. This government institution is based in the administrative district of North Rhine-Westphalia, and has departments for pathology, serology, bacteriology, virology and molecular biology for the diagnosis of animal diseases. It also investigates food and feed quality, and performs pathogen detection in foodstuff. Dr Claudia Bunzenthal from the molecular biology department and her co-workers are also involved in diagnosing other viruses, such as...

Automated High-Throughput Plasmid Purification

Eppendorf Perfectprep® Plasmid 384 Kit on a Tecan Freedom Nucleic Acid Sample Preparation Workstation

For isolating plasmid DNA, various methods are available today, ranging from traditional manual alcohol precipitation to kit-based vacuum extrac-tion procedures performed in 96 well format. However, when it comes to high sample numbers further miniaturization is required to increase throughput and reduce costs per sample. The Eppendorf Perfectprep® Plasmid 384 Kit provides a suitable format for real high throughput automa-tion.

The Perfectprep® Plasmid 384 Kit is a membrane-based system for high-quality plasmid purifica-tion. The combination of ultra-thin membrane with minimized dead volume and...

Automated RNA Extraction

The MACHEREY-NAGEL NucleoSpin® 96 RNA Kit on a Tecan Freedom Nucleic Acid Sample Preparation Workstation

a pivotal role in operation of high-throughput gene expression profiling. However, throughput, quality and quantity of total RNA prepared are often the limiting steps for downstream genetic analysis. The Tecan Freedom Sample Preparation Work-station provides a highly flexible platform for the NucleoSpin® 96 RNA Kit from MACHEREY-NAGEL. The combined use of the Tecan vacuum filtration option (Te-VacS) and the integrated robotic ma-nipulator (gripper tool) allows fully automated total RNA extraction in a 96-well format. More-over, with the use of appropriate storage mod-ules within the Sample Preparation Workstation, multiple batches of 96 samples can be isolated without any manual user interaction.
Purified RNA can be used directly for...

Automated Sequencing Reaction Clean-up

The Promega Wizard® MagneSil™ Sequencing Reaction Clean-Up System on a Tecan Freedom Nucleic Acid Sample Preparation Workstation

ABI PRISM® BigDye™ terminator chemistry is the method of choice for automated, high-throughput DNA sequencing today. However, certain sequencing reaction components interfere with data collection if not removed prior to analysis. Promega has developed a magnetic-based nucleic acid purification technology ideally suited for plasmid DNA. In the Wizard® MagneSil™ Sequencing Reaction Clean-Up System, paramagnetic particles are used for high-throughput purification of BigDye™ terminator DNA sequencing reactions. When automated on the Tecan Freedom Sample Preparation Workstation no user intervention is required for the entire purification process, from the time the 96-well amplification plate is placed on the instrument’s worktable until the samples are loaded onto the DNA sequencer. Sequence quality is similar to manual methods and offers the distinct advantage of an automated protocol.

The Promega Wizard® MagneSil™ Sequencing Clean-Up System is designed to purify BigDye™ Terminator sequencing reaction extension products. The entire process has been designed to...

Compound Dissolution and Reformatting Factory

Automated dissolution, reformatting and storage of compounds

Time is critical – every single day is important, but the requirements for quality and reliability are continually increasing. Scientists need to be able to obtain compounds on demand and in specific patterns, very quickly. Pharmaceutical and biotech companies often have large compound stores and automated dissolution and reformatting platforms. However, these systems are mainly designed and used to support high throughput screening as part of a daily routine, and handling of small batches or individual compounds does not fit ideally into this workflow. For these reasons, Tecan and REMP have designed the Automated Dissolution and Reformatting Factory that integrates the Freedom EVO® liquid  handling workstation with the REMP Small-Size Store™ (SSS).

During a typical dissolution, reformatting and storage workflow, defined amounts of compounds are transferred into vials, which can either be barcode-labelled or...

Nucleic Acid Sample Preparation

Genomic DNA from buccal cells with ChargeSwitch® Technology

Driven by the need for increased sample throughput for genome sequencing projects and diagnostic applications, Tecan has developed a range of automated solutions for nucleic acid purification kits which allows fully automated processing on the tecan robots, with a long walk-away time. Isolation of high-quality DNA is a prerequisite for success in any molecular biology assay. In the coupling of Tecan robotic platforms with ChargeSwitch® Technology, researchers can confidently leave their robotic systems and concentrate their efforts elsewhere. ChargeSwitch® Technology delivers high-yield and high-purity nucleic acid in a rapid and...

Detection of a SNP in the Adenosine A2a receptor gene (ADORA2A) using fluorescence polarization of templatedirected

dye-terminator incorporation (FP-TDI)

Except for identical twins or clones, no two organisms are genetically identical. There is a large range of genetic variation between two members of a species. In humans, two unrelated genomes are estimated to vary between 1 in 500 - 1000 base pairs. Given an estimated size of the human genome of 3 x 109, then there are approximately 3 x 106 DNA sequences that are non-identical between two unrelated individuals. Most of the variations occur as Single Nucleotide Polymorphisms (SNPs). These variants are the substitution of a single base in the DNA sequence for another, such as a C being replaced with a T (or U). SNs are hypothesized to account for much of the genetic variation between individuals and are thought to affect processing of pharmacological agents and certain SNPs may confer higher risk of developing complex genetic diseases, such as, cancer, hypertension, diabetes etc. Although SNPs are numerous, genotyping of this form of genetic polymorphism has been difficult in many cases. One recently described method of enotyping SNPs, FP-TDI (Fluorescence Polarization Template directed Dye Incorporation) is...

Supporting a variety of ELISA testing kits on a single automated platform

Freedom EVOware®

Freedom EVOsim™ – The tool for optimizing scripts and remote monitoring

Freedom EVOware not only controls the Freedom EVO®, but also Freedom EVOsim, a realistic real-time simulation of the Freedom EVO which speeds up script optimisation without having to connect to an  instrument. EVOsim can run on the same PC as EVOware or remotely, and uses innovative technology to make EVOware appear connected to a Freedom EVO. Freedom EVOsim can also be used to monitor a Freedom EVO1 running under Freedom EVOware remotely. Freedom EVOsim uses standard TCP/IP technology to...

Fully automated In Situ Hybridization

High-throughput In Situ Hybridization with the GenePaint system

In situ hybridization is a technique that is used to determine gene expression patterns by localising mRNA transcripts in cells and tissues. The cellular resolution of ISH is by far larger than that of RNA blotting, differential display and microarrays, techniques that are also widely used for gene expression analysis. The reason is that, for in situ hybridization, tissues are not dissected for extraction of RNA, but mRNA transcripts are localised in intact tissue sections, thereby visualising gene expression in individual cells or clusters of cells within their tissue context. The signal obtained shows where a gene is ‘expressed’, i.e. where it is active. Gene activity depends on promoters, which are DNA sequences that drive the transcription of genes into mRNA. Thus, in situ hybridization is a powerful tool to determine the cell- and tissue-specific activity of promoters. In addition, genes and their products are usually functional in the cells where they are expressed. Therefore, in situ hybridization is also used to...

Automated IHC analysis of tissues on slides

Automated IHC analysis of swine fever, TSE, BSE and scrapie on paraffin-embedded and frozen tissues

Immunohistochemistry (IHC) is a detection technique used not only for research, but also diagnostics of animal epidemics. The method is established in the international and EU regulations for the diagnosis of notifiable animal epidemics. For the diagnosis of classical swine fever (KSP), the use of the fluorescent antibody test (FAT) on cryosections is laid down in the OIE Manual (literature) and in the EU diagnostics manual (1, 4). For the diagnosis of transmissible spongiform encephalopathy (TSE), bovine spongiform encephalopathy (BSE) and scrapie, the use of immunohistochemical detection methods (IHC) on formalin-fixed tissue is required according to...

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.

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.

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.

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.

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).

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.

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.

Accelerate Drug Discovery with Hewlett-Packard Direct Digital Titration
Christie Dudenhoefer (christie.dudenhoefer@hp.com), Jeff Nielsen, Matthew Still, Joshua Yu, Dave Ochs

Introduction

The Hewlett-Packard D300 Digital Dispenser adds breakthrough capability to the dose-response workflow by eliminating the need for serial dilution and enabling researchers to quickly complete complex dose response experiments at the bench. HP technology dispenses picoliter to microliter volumes of compounds in DMSO solution directly into 384, 96, 48, 24 and 12 well assay microplates. Doses are built up as picoliter drops of stock compound are added to the wells.

HP’s Digital Dispenser Software and its simple user interface (UI) is the gateway to easily set up plate layouts.

This Tech Note covers the following aspects of the elegant and powerful software driving HP dispensing technology:

I. Software basics
II. Standard plate layouts
III. Special plate layouts

 1. Randomized layouts
 2. Finely spaced dose creation
 3. Drug‐drug interaction studies
 4. Multi‐plate dispensing
 5. Normalization

...

Introduction

In this application note, we describe the use of peptide microarrays from JPT Peptide Technologies to characterize antibody reactivity in healthy human plasma towards an
antigen of the Epstein-Barr virus using Tecan array technology (HS Pro hybridization stations and PowerScanner™). We explain how detailed mapping of humoral immune response can be performed, and present some of the conclusions drawn from the study.

Microarrays are powerful tools used to perform multi-analyte assays efficiently in a highly parallel manner. The initial development of array technologies was driven mainly by the advances in DNA array technologies. However, soon afterwards other analytes, such as carbohydrates, lipids, proteins and peptides, were immobilized.

Since then, peptide microarrays have demonstrated usefulness in fields ranging from substrate profiling of orphan enzymes1,2 to profiling of humoral immune response in plasma and serum from multiple species, as well as in other biological fluids. The latter use led to the identification of correlates for the protective mechanism of an HIV vaccine in the RV144 and other trials3,4,5. Peptide microarrays facilitate the presentation of random, as well as knowledge-based libraries. They also make the display of an entire protein as overlapping peptides possible (Figure 1), including sequence polymorphism and a wide range of defined post-translational modifications.

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The ability to monitor large changes in large numbers of miRNAs simultaneously is a key factor in understanding miRNA function.Exiqon has therefore developed miRCURYTM LNA Arrays for large-scale investigation of miRNA expression.The Tecan series of HS ProTM hybridization stations (Tecan, Austria) enables full automation of microarray hybridization experiments on microarray slides by producing uniform and reproducible results with minimal handling of solutions and slides.

A new version of the whole-genome microarray has recently been developed at the Wellcome Trust Sanger Institute.For the first phase of this project, mor than 500 hybridizations have been processed on the HS 4800™ hybridization station in six months, with an estimated success rate of more than 80%.

This application note details the use of the Tecan HS 4800™ Hybridization Station and proves the suitability of the instrument for ISH applications, including all hybridization, washing and immunostaining steps.

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.

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

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...

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....

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...

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

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.

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.

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.

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...

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

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

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

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

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.

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”.

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.

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 F500 filter based multimode detection system. Tecan’s Infinite F500 offers an easy-to-use and flexible way of accessing fluorescence polarization data.

This technical note describes the successful implementation of HTRF® measurements on the Tecan Infinite® F500 filter based multifunctional reader. The Infinite F500 was proven to be a very well-suited detection platform regarding sensitivity and dynamic range of the investigated IL-1β detection - HTRF®assay kit.

This technical note describes the successful performance of Invitrogen´s PolarScreen™ Vitamin D Receptor Competitor Assay, Red on the Tecan Infinite® F500 filter based multimode detection system. In regard to the obtained data the instrument is perfectly performing according to the given assay requirements.

The data obtained from the measurements reveal the correct separation of blue and green luminescent signals. The calculated values are comparable to those given by the kit manufacturer, PerkinElmer. They clearly underline the positive signal of the transfected and the negative signal of the non-transfected cells. Only slight variations between the use of a 96 well or 384 well microplates were observed, thus making the assay ready for higher throughput using the Tecan Infinite® F500 filter-based microplate reader.

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

Optimizing the FI bottom sensitivity in the Infinite F500 multimode reader

Optimizing the luminescence sensitivity in the Infinite F500 multimode reader

Implementation on Tecan’s Infinite® F500 multimode reader

The Infinite® F500 is Tecan´s most sensitive filter-based multimode microplate reader and has been granted LanthaScreen® Certified Plus status by Invitrogen.
In this technical note we describe the instrument settings on the basis of experiments with the LanthaScreen® TR-FRET Control Kit on Tecan´s Infinite F500 multifunctional detection system.

Detection of human immunoglobulin G (IgG) using the Infinite M1000 PRO

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.

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

Sensitivity, uniformity and crosstalk of AlphaScreen measurements in the Infinite M1000 PRO

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

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

Optimizing the FI top 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

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.

Using Tecan’s Infinite® F50 ELISA reader and the Greiner Bio-One Saliva Quantification Kit

This application note describes the outcome of a successful evaluation study of Tecan’s Infinite F50 microplate reader in combination with Tecan’s Magellan™ data analysis software, for the fast detection of saliva samples using the Saliva Quantification Kit from Greiner Bio-One.

Automated sample preparation for AB SCIEX 3200 QTRAP® system using Tecan Freedom EVO® liquid handling workstation

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.

Fast and reliable extraction of plant DNA using the NucleoMag® 96 Plant kit on a
Freedom EVO® platform equipped with a MultiChannel Arm™ 384 (MCA 384)

Introduction

Plant research is often geared towards crop improvement, and therefore focuses on yield and robustness to pathogens and other stress factors, such as heat or draught. Common applications include TILLING (Targeting Induced Local Lesions IN Genomes) and the creation of genetically modified species, as well as traditional breeding technologies. In all cases, the breeding success must be confirmed not only by phenotyping, but also by genotyping, creating a need for high throughput genomic DNA extractions. The  analysis of plant material in food diagnostics has similar requirements, for
example where the presence or absence of genetic modifications needs to be verified. DNA extraction from plant material is therefore an integral step in both plant research
and food analysis.

MACHEREY-NAGEL has developed the NucleoMag 96 Plant kit to meet the demand for fast and homogeneous extraction of high quality DNA from a variety of plants and fungi. This magnetic bead-based extraction process delivers high quality DNA and keeps the workflow very flexible with regard to scalability (the amount of starting material) and sample numbers.
Tecan and MACHERY-NAGEL have joined forces to provide a flexible automated solution for the isolation of genomic plant DNA without compromising yield or purity. After the initial homogenization of the plant material, the workflow can be completely automated on a Freedom EVO sample preparation workstation, reducing the risk of contamination, carry-over and manual errors to a minimum. Sample tracking further increases both sample and overall process security.

...

Tissue gDNA extraction with MACHEREY-NAGEL’s NucleoMag® 96 Tissue kit and the Freedom EVO® workstation

Rapid extraction of genomic DNA from a variety of food and feed samples with NucleoSpin® 8/96 Food columns and the Freedom EVO®

Purification of large numbers of blood samples represents a serious bottleneck in sample processing for genotyping or general screening projects. Furthermore reliability, process control and the avoidance of cross-contamination are major issues for the purification of gDNA.

Purification of large numbers of blood samples represents a serious bottleneck in sample processing for genotyping or general screening projects, and reliability, process control and the avoidance of cross-contamination are major issues for the extraction of gDNA.

Fast and reliable DNA extraction from plants with NucleoMag® 96 Plant from MACHEREYNAGEL on a Freedom EVO® platform.

Avian influenza is a bird disease caused by the highly pathogenic influenza virus. Influenza viruses are inherently unstable and, as they lack a genetic proof-reading mechanism, small errors that occur when the virus copies itself go undetected and uncorrected. Specific mutations and evolution in influenza viruses cannot be predicted...

High-throughput plant DNA isolation technology plays a pivotal role in large plant genotyping projects (population studies, plant breeding investigations and genetic modification of organ-isms). However, the throughput, quality and quantity of total DNA prepared are often the limiting steps for downstream genetic analysis...

Art & Science of Liquid Handling

MINIPREP Series Robotic Sample Processors

Tecan’s Gemini software for applications development incorporates a unique new tool to help developers of robotic applications manage these factors. The Liquid Class Database allows the management of all factors that influence liquid handling in an organized manner. Pre-programmed liquid classes are provided. The user can copy, rename, then edit the default settings to create new custom liquid classes. The Liquid Class Database will not only save you time when developing your first robot application, but it will allow you to apply your experience quickly to subsequent methods that you may develop.

This note provides fundamental concepts that will help you optimize your automated liquid handling applications and includes the following topics:

• Pipetting Modes
• Z-Values
• Liquid Level Detection
• Airgaps
• Aspiration and Dispense Speeds
• Wash Procedures
• Additional Precision and Accuracy Considerations
• Default Settings and Procedures

A Semi-automated Method Using NucleoSpin®

Extract Kit on Tecan MiniPrep MolBio Separation System

Several methods are available for purifying PCR fragments from the reaction mixtures. Macherey-Nagel’s NucleoSpin® Robot-96 Extract Kits provide fast, economical means of processing up to 96 samples simultaneously, typically within 74 minutes. The kit provides reagents and consumables for purification up to 15 μg DNA per well. Primers, primer-dimers, nucleotides, salts and polymerase are removed effectively. The PCR products are suitable for standard molecular biology applications like DNA sequencing, cloning or microarray technology. [Also see Macherey-Nagel references for kit descriptions, Cat. No. 740707.2 or 740707.4.] Tecan describes a semi-automated purification process based on...

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

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...

Introduction

In this application note, we describe the use of peptide microarrays from JPT Peptide Technologies to characterize antibody reactivity in healthy human plasma towards an
antigen of the Epstein-Barr virus using Tecan array technology (HS Pro hybridization stations and PowerScanner™). We explain how detailed mapping of humoral immune response can be performed, and present some of the conclusions drawn from the study.

Microarrays are powerful tools used to perform multi-analyte assays efficiently in a highly parallel manner. The initial development of array technologies was driven mainly by the advances in DNA array technologies. However, soon afterwards other analytes, such as carbohydrates, lipids, proteins and peptides, were immobilized.

Since then, peptide microarrays have demonstrated usefulness in fields ranging from substrate profiling of orphan enzymes1,2 to profiling of humoral immune response in plasma and serum from multiple species, as well as in other biological fluids. The latter use led to the identification of correlates for the protective mechanism of an HIV vaccine in the RV144 and other trials3,4,5. Peptide microarrays facilitate the presentation of random, as well as knowledge-based libraries. They also make the display of an entire protein as overlapping peptides possible (Figure 1), including sequence polymorphism and a wide range of defined post-translational modifications.

...

Fast multiple plate replication

Use of 384-channel parallel dispensing

The replication of microliter or nanoliter aliquots of compound solutions in DMSO with low well-to-well variability of the aliquot volume is a de-manding liquid handling task. The challenge is set forth by several prerequisites and require-ments:

• Because the final DMSO concentration in the assay volume has to be kept low, compound aliquots for HTS have a very small volume and are highly concentrated.
• DMSO is highly hygroscopic, but has to be kept nearly water-free to enable freezing of the compound solution at -20°C.
• To minimize the uptake of water during the aliquoting process, one replication cycle should be used to prepare multiple aliquots in high-density plates for multiple HTS projects.
• To achieve high-throughput in the plate repli-cation process for preparation of aliquots in 384 or 1536-well plates, parallel processing of (at least) 384 wells is crucial.

To cope with these demands, we have set up a high-throughput plate replication system for preparation of up to 80 replicas out of one source plate in one replication cycle. The num-ber of aliquots prepared in one replication cycle is adjusted to...

Blood pooling

Te-PoolSafe™ option for automation of blood pooling ensures safe PCR diagnostics

The constant availability of blood is essential for saving lives, not only of people who suffer serious accidental injury, but also of patients undergoing surgery – a thoracic operation, for example, can require 20-30 units of blood. Blood is truly a critical commodity because, at present, there is no artificial alternative to human blood collected from blood donors. To compound this problem, blood donors, who are already in short supply, undergo a rigorous selection process, so every single donation is precious and therefore cannot be wasted by...

PCR - polymerase chain reaction
ELISA - Enzyme-Linked Immunosorbent Assay

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 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...

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...

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) 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...

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,...