Attana has several Quartz Crystal Microbalance (QCM) based instruments and applications, all catering to crucial needs and developed in close collaboration with industry and universities. Examples include kinetic analysis on cell surfaces, candidate selection screening, aggregation characterization, epitope mapping and concentration determination of biopharmaceuticals.

Introduction

Commercially developed by Attana AB and distributed by Tecan for scientific use only, Attana’s quartz crystal microbalance (QCM) technology is a non-optical measurement technique that enables the real-time, label-free characterization of molecular binding kinetics of biomolecules, such as antibodies, antibody fragments, or single chain antibodies to an immobilized receptor or cell on the sensor surface, with a variety of sensor materials available to cover a broad range of applications including working with macromolecules, viruses, bacteria, and immobilized cells.

Biochemical interaction analyses

The Attana 200® instrument enables determination of specificity, target affinity, and binding kinetics for a wide variety of biomolecules. By enabling this characterization to be performed in crude biological samples or sera, The Attana 200® instrument enables determination of specificity, target affinity, and binding kinetics for a wide variety of biomolecules. By enabling this characterization to be performed in crude biological samples or sera, Attana’s QCM technology provides researchers with more biologically relevant information, as well as eliminating the need for sample purification.This allows researchers to select suitable biomolecules based on kinetic parameters – such as preferential “off-rates” – rather than just target-affinity data using concentration dependent techniques such as ELISA.

Cell-based interaction analyses

Cell-based interaction analyses The Attana Cell™ 200 can perform real-time, label-free characterization of molecular interactions in either a biochemical or cellular environments (using immobilized cells), enabling the investigation of the dynamic properties and concentration and time dependent nature of binding. This allows researchers to compare the binding characteristics of biomolecules, in the more biologically complex context of crude samples or sera, on the cell surface.

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Jeff Nielsen (jeff.nielsen@hp.com), Ken Ward, Christie Dudenhoefer, David Ochs, Joshua Yu, and Kevin F. Peters

Introduction:

HP has been the worldwide leader in inkjet technology for 25 years, and HP printers reliably dispense billions of picoliter droplets per day. The HP T8 Dispensehead is built on a silicon wafer using HP inkjet photolithography to enable precise, accurate drop generation and placement. The single-use dispensehead utilizes 22 high-frequency nozzles so that picoliter to microliter volumes are dispensed in under a second...

Digital Dispensing Overview:

Digital dispensing enables dose-response analyses without the use of serial dilution. This direct-dilution process generates a dose directly from stock solution by ejecting the appropriate number of picoliter droplets into each well. This means each dose is independent of every other well. Intermediate and serial dilution steps can be eliminated, enabling simplified workflows. Dispensing is accomplished by jetting droplets into either dry or previous filled wells...

Method:

The ease and flexibility with which plate dispensing protocols can be created and executed with a non-contact digitaldispensing technology enables certain types of experiments that would not otherwise be practical...

Results: Drug-Drug Interaction:

The top portion of Figure 7 shows software screenshots from a typical co-titration, with each color representing a drug, and each level representing a concentration. The total time to program this protocol and dispense this plate is five minutes...

Outstanding Precision:

Key to the performance of the HP D300 Digital Dispenser is excellent precision over a wide dynamic range. Three assay plates were created to assess the instrument precision across five orders of magnitude: one high volume absorbance-based plate and two fluorescence-based plates. Four test volumes were dispensed per plate and ten replicate wells were dispensed per Dispensehead at each volume...

Conclusion:

Direct dilution using HP picoliter digital dispensing technology is a replacement for serial dilution. Compound usage can be reduced by 90%, while reliability and speed are improved with simplified workflows. Non-contact dispensing of droplets directly into assay wells make it practical to routinely perform drug-drug interaction experiments...

Tracy Worzella, Gediminas Vidugiris, Jolanta Vidugiriene, Jacquelyn Turri, Hicham Zegzouti, Lyndsey Helley, Jessica Merlino, Michael Reitman
Promega Corporation, Madison WI; Tecan Group AG, Männedorf, Switzerland

1. Abstract

Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activity. While automation is necessary to screen and identify putative inhibitors, it can also provide benefit for followup characterization studies where throughput is not a primary concern. Smaller, more cost-effective platforms are now available that offer the user programming flexibility and ease of use to carry out the tasks needed to profile their compounds of interest in-house. Combined with sensitive reagents, these instruments comprise a semi-automated workflow for lower throughput kinase profiling applications...

2. Kinase Enzyme Systems and the ADP-Glo™ Kinase
    Assay...

3. Tecan HP D300 Digital Dispenser...

4. Small Footprint Instrumentation Fits Into The Kinase
    Profiling Workflow...

5. Comparison of Gefitinib Titration Method With EGFR
    Kinase...

6. Lipid Kinase Profiling: Rapid Profiling of 11 Kinases
    with 8 Known Inhibitors...

7. Profiling Gefitinib Against On-Target and Off-Target
    Kinases...

8. Conclusions

• Small footprint, stand alone instrumentation for compound dispensing, reagent dispensing, and detection can be used together to profile on-target and off-target effects on kinase activity.

• Out-of-the-box optimized Kinase Enzyme Systems enabled rapid profiling of kinases of interest without the need to configure assay conditions before experiments were performed. Kinase Profiling Systems, which include kinases and substrates in 8-well strip format, will soon be available.

• The broad dispense range of the HP D300 enabled potency determination experiments to be performed using one source concentration of compound to assemble titration curves, with minimal requirements to incorporate an intermediate dilution.

• Performing dose response testing with a 24-point single replicate titration curve provided equivalent results to a more conventional multi-replicate method, enabling savings on compound, reaction and assay components.

• Setting up profiling of lipid kinases from different PI3K and PI4K families was rapid, with results showing robust assay performance.

• One assay plate was used to simultaneously profile 16 kinases against Gefitinib, with results showing selective inhibition of the target kinase, EGFR, with minimal off-target effects.

The HP D300 Dispenser is an easy to use device for rapid contact free dispensing of DMSO solutions with minimal prime volumes. The device dispenses single droplets of only 13 picoliters (!) and bigger volumes are digitally build up from multiple droplets. The remarkably small volume that is transferred with a single shot allows the direct dispensing of stock solutions into assay plates, spanning a wide range of concentrations of the test compound. We routinely use the instrument for the rapid generation of titrations e.g. of substrates for Km determinations or of inhibitors for IC50 determinations or for active site titrations in the context of assay development for in vitro biochemical assays. Furthermore, the small droplet volume allows the direct dispensing of DMSO solutions onto live cells without compromising cell viability due to the dispensing process. The device is robust with a small footprint as a standalone device and the control software is intuitive and easy to use.

Digital Dispensing

Digital dispensing provides the ability to form dose-response analyses without the use of serial dilution, instead forming the series of doses by directly placing the appropriate number of picoliter droplets into each well. This greatly simplifies work flows by eliminating intermediate steps in forming dose series. This is also called direct dilution since every well is given its dose directly from the stock, so that the dose in every well is independent of the dose in any other well...

Picoliter Dispensing with HP Inkjet

HP has been the leader in inkjet technology for 25 years, and HP printers reliably dispense picoliter droplets billions of times per day around the world. Now this mature technology has been leveraged for pharmaceutical research...

Digital Advantages

A series of direct dose-response curves were developed for a cell-based influenza fluorescence assay with a Z' of 0.85. Comparisons were made between IC50 values determined from eight point triplicates, 16 point singlets and 24 point singlets. In addition, known information about IC50 values was used to develop a series of targeted doses, with doses densely spaced near the IC50 value and sparsely spaced near the plateaus. While traditional serial dilutions are limited to evenly spaced doses, targeted designs are easily created using the HP Digital Dispenser software as shown in Figure 5...

Summary of Results

A series of direct dose-response curves were developed for a cell-based influenza fluorescence assay with a Z' of 0.85. Comparisons were made between IC50 values determined from eight point triplicates, 16 point singlets and 24 point singlets. In addition, known information about IC50 values was used to develop a series of targeted doses, with doses densely spaced near the IC50 value and sparsely spaced near the plateaus. While traditional serial dilutions are limited to evenly spaced doses, targeted designs are easily created using the HP Digital Dispenser software as shown in Figure 5. Table 1. IC50 values and standard errors showing D300 precision. IC50 values and their standard error were determined in XLFit using a four parameter model for the three compounds as shown in Figure 7 and Table 1. Note that the standard errors of the 8 point triplicate designs are larger than for the singlet designs, showing the improvement of fine spacing while using fewer wells. Also note the excellent reproducibility of pIC50 values for various experimental designs for all three compounds over a wide range of IC50 values on the HP D300 Digital Dispenser...

Conclusions

Direct dilution using HP picoliter digital dispensing technology is a simple replacement for tedious and error-prone serial dilution. Compound usage can be reduced by 90%, while reliability and speed are greatly improved with simplified workflows. And as shown in this poster, dispensing ultrasmall droplets directly into assay wells enables new scientific approaches to the design of dose-response analyses. These advantages include finely spaced and targeted doses...

Drawbacks of traditional serial dilutions

The traditional serial dilution process for IC50 determinations of small molecules has numerous drawbacks, including:

• being limited to working in the microliter range;
• superfluous compound consumption;
• high risk of (cross-)contamination;
• inter-well dependencies;
• edge effects;
• limited data accuracy;
• poor inter-operator and inter-laboratory reproducibility;
• excessive consumables usage;
• high consumption of tips and bulk reagents.

Serial dilutions also typically require a tremendous effort in upfront preparation, either to manually calculate and set up a dilution, or to program automated processes...

Introduction

Researchers are increasingly turning to 3D cell culture techniques for their studies, due to the improved physiological relevance of the cellular environment. These cultures promise to yield responses to stimuli – such as the addition of a drug candidate – that are more similar to those seen in animal models or humans. TAP Biosystems has developed a novel 3D cell culture technique, RAFT (Real Architecture for 3D Tissue), which allows researchers to culture cell types of their choice in an in vivo like environment. The technology uses the most abundant matrix protein in the body, type I collagen. The RAFT process raises the collagen concentration to physiological levels quickly and reproducibly while maintaining high cell viability.

TAP Biosystems and Tecan have worked together to develop an automated process that enables researchers to increase throughput, facilitate mixing procedures and enhance reliability while maintaining cell viability, reproducibility, experimental versatility and analytical flexibility...

Introduction

Oncotest maintains a unique repository of > 300 tumor xenograft models representing all major tumor histotypes (non-small cell lung cancer/ NSCLC, pancreatic, prostate, colon, gastric, breast, ovarian and renal cancers, melanomas and sarcomas) as well as niche tumors (pleuramesothelioma, bladder, and head and neck cancers). After evaluating many 3D cell culture platforms, Oncotest has chosen Alvetex®Scaffold 96 because of its simplicity, low cost of adoption and flexibility to study tumour cell cytotoxicity as well as mechanism of drug action at the translational and post-transcriptional levels...

Introduction

Researchers are increasingly turning to 3D cell culture techniques for their studies, due to the improved physiological relevance of the cellular environment. These cultures promise to yield responses to stimuli – such as the addition of a drug candidate – that are more similar to those seen in animal models or humans. TAP Biosystems has developed a novel 3D cell culture technique, RAFT (Real Architecture for 3D Tissue), which allows researchers to culture cell types of their choice in an in vivo like environment. The technology uses the most abundant matrix protein in the body, type I collagen. The RAFT process raises the collagen concentration to physiological levels quickly and reproducibly while maintaining high cell viability.

TAP Biosystems and Tecan have worked together to develop an automated process that enables researchers to increase throughput, facilitate mixing procedures and enhance reliability while maintaining cell viability, reproducibility, experimental versatility and analytical flexibility...

Introduction

Oncotest maintains a unique repository of > 300 tumor xenograft models representing all major tumor histotypes (non-small cell lung cancer/ NSCLC, pancreatic, prostate, colon, gastric, breast, ovarian and renal cancers, melanomas and sarcomas) as well as niche tumors (pleuramesothelioma, bladder, and head and neck cancers). After evaluating many 3D cell culture platforms, Oncotest has chosen Alvetex®Scaffold 96 because of its simplicity, low cost of adoption and flexibility to study tumour cell cytotoxicity as well as mechanism of drug action at the translational and post-transcriptional levels...

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.

Here we demonstrate the use of a non-activity-based cytotoxicity probe, CellTox™ Green Dye, that can be added at the beginning of the experiment and employed in real time for the kinetic determination of cytotoxicity. We used the Tecan HP D300 Digital Dispenser for non-contact dispensing of test compound, and the Tecan Infinite® 200 PRO with GCM™ for kinetic measurement of cytotoxicity over 72 hours...

Abstract

Growth analysis of bacteria and yeast via OD 600 nm measurements is key to many different research areas, and used to be a time-consuming and laborintensive procedure. Instead of shuttling samples or microplates between the incubator, Tecan now provides control of all essential growth conditions within the Infinite 200 PRO microplate reader, including temperature, O2 (0.5-21 %), CO2 (0-10 %) and humidity stabilization.

Introduction

In this collaborative study, Tecan, Thermo Scientific and partners have analyzed the growth of the microaerophilic human pathogen and class-I carcinogen H. pylori over a time period of 28 hours. As a microaerophilic organism, H. pylori needs hypoxic  conditions to colonize the mucosa of humans, where it causes stomach disorders and cancer. 

Methods

Bacteria were incubated and measured simultaneously inside the Infinite 200 PRO multimode microplate reader at 37 °C, 10 % CO2 and varying O2 (21 % (control), 5 % and 10 %).
The proliferation was monitored by detecting the absorbance signal of the samples at 600 nm and the fluorescent signal of green fluorescent protein (GFP) transformed H.  pylori.

This Poster 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®
certifi cation program criteria and was successfully validated by Invitrogen as
“LanthaScreen® Certified”.

Transdermal drug delivery has extensively grown in the past decades. Advantages of transdermal application dosage forms like plasters in certain drug families have been proved. Efforts in drug research have been devoted to find a useful model for predicting the skin penetration of new molecules. The parallel artificial membrane permeability assay (PAMPA)1 as a passive permeation screen method can be used for the ADME screening of molecules.

Our purpose is to develop an effective skin PAMPA model using similar ingredients are present in the real human skin.

Avidity determination of different anti-EBV-IgGs against single antigens using the ProfoBlot.

Abstract

The Infinite M1000 PRO is the latest addition to Tecan’s high-end microplate reader  portfolio. In addition to the established reading modes, it now also features a high-end module for AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay), a bead-based screening technology developed for fast, reliable and cost-effective detection of biomolecular interactions. To achieve uncompromised performance for Alpha-based applications, the Infinite M1000 PRO uses a combination of a powerful laser light source and dedicated emission filters for AlphaScreen and AlphaLISA assays. The instrument’s luminescence detector is used for ultra-sensitive detection of the Alpha signal. Along with the advanced optics module, the Infinite M1000 PRO offers an ingenious real-time temperature correction (TC) function for Alpha-based assays that serves to compensate for sample temperature variations across the microplate.
In addition to the evaluation – using PerkinElmer’s AlphaScreen Omnibeads™ chemistry – of common assay quality parameters such as sensitivity, uniformity and crosstalk,
the Infinite M1000 PRO has been tested in regard to the P-Tyr-100 AlphaScreen and human immunoglobulin G (IgG) AlphaLISA assay. The Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, has run a pilot screen based on the ChemBioNet library, to test the Infinite M1000 PRO in an academic screening setting.

This Poster describes the implementation of HTRF® (Homogeneous Time Resolved Fluorescence;Cisbio international, France) assay systems on the Infinite® M1000, Tecan’s new high end multimode monochromator-based detection system. For the HTRF® validation of the Infinite M1000 three different HTRF® assays have been used, the HTRF® Reader Control Kit, the HTRF® cAMP assay and the HTRF® TNFa assay

Evaluation of an Automated Liquid Handling System and Application Software for Streamlined Processing of Forensic DNA Samples Using the Tecan Freedom EVO® 150 and Applied Biosystems 7500 Real Time PCR System and 3130xl Genetic Analyzer

This presentation summarizes the work performed to develop and evaluate an automated liquid handling system for DANN quantification and STR profiling using the Applied Biosystems Quantifiler® NDA Quantification Kits and AmpFLSTR® PCR Amplification Kits. The hardware for the automated system consists of a Tecan Freedom EVO® 150 for liquid handling, the Applied Biosystems 7500 Real Time PCR System for DNA quantification, an Applied Biosystems GeneAmp® PCR System 97000 for STR amplification and an Applied Biosystems 3130xl Genetic Analyser for detection of the amplified STR markers. Specialized application software, HID EVOlution™ Software, was used to track samples throughout the process and facilitate transfer of information from one instrument to another.

Pipetting methods and liquid classes were developed by Tecan automation specialists based on the specific components and requirements of each Applied Biosystems kit. All methods and classes were individually tested and evaluated on the Freedom EVO® 150 prior to testing in the completed system. Cross contamination studies were performed
to confirm the ability of the Freedom EVO® 150 to consistently assemble reactions without contamination. Testing of individual software components was performed through software development. Evaluation of the entire automated system consisted of processing mock forensic sample DNA extracts through DNA quantitation, DNA normalization, PCR amplification and...

Automated High Throughput Transfection of Primary Cells

Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling platform and include all the necessary components and...

Automation of QPCR and PCR methods for forensic casework samples on the Tecan Freedom EVO®

In the last decade, DANN analysis for human identification in forensics has led to tremendous improvements in the resolution of criminal cases. Automated systems are already in use to feed convicted offender databanks in various countries. Concerns are different in processing crime scene samples: variability of sample types (blood, semen, hair, cells, etc.), sample to sample contamination, etc. Creation of national DNA databanks has led to a dramatic increase in the number of crime scene samples submitted to forensic laboratories. Consequently, it has become obvious that...

Freedom EVO® | Forensics

Complete automation of Promega DNA IQ™ System for DNA extraction in casework and databasing laboratories

• Recovery of DNA – equivalent or better than manual and other automated implementations of DNA IQ™ System
• Can handle a wide variety of sample types including most commonly encountered in forensic casework, and can start with samples either in plates or tubes
• Using disposable tips, 96 samples can be run in about 1.5 hrs on a 8 tip instrument and in about 2.25 hrs on a 4 tip instrument
• Purified DNA can be eluted in volumes ranging from 40 μL to 100 μL
• Unique ease-of-use software GUI guides through system setup
• Reuse of disposable tips helps lowering costs and minimizes risks of cross contamination

Optimizing bacterial growth studies on the Infinite® 200 PRO multimode reader platform using permanent shaking and heatingThe Infinite F500 is Tecan’s most sensitive filter-based multimode reader. It is designed as a multifunctional and modular detection system that supports a broad range of detection modes, including absorbance, fluorescence (top/bottom), luminescence (flash/glow/multicolor), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF) and the combination of the latter two techniques, TR-FRET (timeresolved fluorescence resonance energy transfer) [1-2].

This poster describes the successful implementation of BellBrook Labs’ Transcreener ADP FP assay and Transcreener ADP2 FI assay on Tecan’s filter-based Infinite F500 and the premium Quad4 Monochromators™-based Infinite M1000 multimode microplate readers. The exemplified measurement data presented on this poster demonstrates an outstanding performance of the Infinite M1000 and the Infinite F500 with both the new fluorescence intensity (FI)-based and the fluorescence polarization (FP)-based Transcreener technology. Optimized instrument settings are stated to achieve optimal assay performance. Experiments were performed at BellBrook Labs, USA and Tecan Austria.

Introduction
Cell-based assays are powerful and versatile tools for experimental design in life science research, and are frequently used for high throughput screening analysis. Cell
adhesion, chemotaxis, cell migration and invasion are essential topics in cancer development and tumor growth. Cell-based assays on micro-porous membranes provide
outstanding in vitro models for drug discovery in research areas such as angiogenesis, neoplasia and inflammation.

Principle of FluoroBlok cell culture inserts
BD Falcon FluoroBlok cell culture inserts allow easy and non-destructive analysis of invasion and migration assays, as well as offering automation compatibility (1). Fluorescently labeled cells inserted into the top chamber migrate through the membrane and are detected by bottom reading fluorescence plate readers and microscopes.
Cells present in the top chamber of the insert are shielded by a light-tight polyethylene terephthalate (PET) membrane that blocks light within the 490-700 nm range. BD Falcon
FluoroBlok cell culture inserts are available in different formats, pore sizes and protein coatings for various applications (1).

Inducing cell migration
In the initial experiments, migration of cells through the FluoroBlok filter was validated by inverted microscopy; however, cell quantification is constrained by its variability, due to cell spreading in the presence of serum, and by the fact that it is very time-consuming. To improve the throughput of this assay, quantification of cell migration was performed by measuring fluorescence intensities in the FluoroBlok bottom chambers using the Infinite M200 PRO multimode microplate reader...

Automatic Cell Line Propagation, Expansion and

Assay Preparation on Tecan® Cellerity™

Cellerity is Tecan’s solution for fully automated culturing of several cell lines in parallel utilizing microplate-sized cell culture flasks and plates. Cellerity was configured to deliver HEK-293 cells in 384 well assay plates ready for compound screening. A series of replicate plate production runs were produced by Cellerity over the course of 22 days. The data generated on cell count, cell viability and functionality demonstrate that Cellerity is able to automatically and reliably deliver cells in the required quality for...

Replication of data sets is necessary for drawing reliable conclusions from microarray experiments, and high sensitivity in the form of high signal-to-noise ratios extends the range of expression levels that can be analyzed in such experiments. Tecan’s LSx00 series of scanners combined with ArrayPro 4.5TM software from MediaCybernetics offers both high throughput and increased sensitivity.

Detection of a SNP in Adenosine A2a receptor gene using fluorescence polarization of

template-directed 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, there are approximately 3 x 106 DNA sequences that are non-identical between two unrelated individuals. Most of the variations of all known polymorphisms occur as Single Nucleotide Polymorphisms (SNPs). These variants are defined as substitution of a single base in the DNA sequence for another, such as a C being replaced with a T (or U) in case of Adenosine A2a receptor gene (ADORA2a). SNPs 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 genotyping SNPs, FP-TDI (Fluorescence Polarization Template directed Dye Incorporation) is simple, non-radioactive and relatively inexpensive. The method is...

Western Blot analysis is used in many key applications in clinical diagnostics including the screening and confirmation of different organisms causing infectious diseases. At the same time, the introduction of the IVD-Directive 98/79/EC is setting new standards for a wide range of tests and assays. Designed to improve safety and reliability in the laboratory, the new generation ProfiBlot 48 is a fully IVD-compliant automated instrument for Western Blot analysis.

Improving quality and throughput of assays for genomic and proteomic research in South Africa. The Centre for Proteomic and Genomic Research (CPGR) has several Tecan instruments for automated processing of multiple batches of microarrays simultaneously, including an HS 4800™ Pro Hybridization Station with the new QuadChamber™ and a HydroFlex™ system.