Tecan Journal Edition 01/2005

Tecan JournalEdition 1 2005

ISSN 1660-5276

Cellerity™ meets customer needs forautomated cell culturepage 6

HS 400™ Hybridization Station makeslight work of genomic DNA patternspage 8

Talk to Tecan’s Customer Supportpage 14

2 G LO B A L N E W S

Tecan Journal 1/2005

Firstly, we would like to take thisopportunity to introduce ThomasBachmann, who joined Tecan in February2005 as the new CEO. Thomas brings awealth of experience in internationalsenior management to his new role andwill be focusing his efforts on employees,customers and investors alike.

Welcome to the “new look” Tecan Journal

“My strong personalcommitment to Tecan isto continue developingexcellent relationshipswith our customers andpartners, and to maintaintheir confidence and

trust. We will focus on issues that areimportant to our customersand aim to win the respectof the life science, diagnosticand pharmaceutical worldsby consistently providingthe solutions and supportthey need.”

Our presence in the Asia-Pacific region has been boosted by the opening of the Tecan office in Beijing. Our newTecan representatives are Mark Wang([email protected]) and BarbaraStobbe ([email protected]).

Distributors in this region recently met atthe Asia Pacific Distributor Sales Meeting2005 in Bangkok. The meeting washosted by Marco Vittoz, Director SalesInternational, and attended by more than20 members of Tecan South East Asia(SEA) distributors and Tecan representa-tives from China and (SEA), as well asnew area managers for India and NewZealand. During the meeting, informationwas shared between Tecan and thedistributors on a personal basis andTecan’s sales and support strategy in theAsia Pacific market was discussed.

Thomas Bachmann Chief Executive Officer(CEO)

Training Mark Wang at Tecan Switzerland and Austria

Asia Pacific Distributor Sales Meeting 2005 in Bangkok

Barbara Stobbe and Mark Wang


DetectionTaking microarrays by stormTecan teams up with Novartis to create an impressive new microarray technologywith the potential for use in novelapplications page 4

Application BiopharmaCellerityTM meets customer needs forautomated cell cultureCellerity, Tecan’s new fully automatedsystem for cell line growth andmaintenance, promises to be the mostaffordable, compact and flexibleinstrument of its kind page 6

Detection/Application BiopharmaHS 400TM Hybridization Station makeslight work of genomic DNA patterns

The HS 400 is used to investigatemolecular changes along differentpathways in hereditary non-polyposiscolorectal cancer page 8

Tecan prepares launch of new hybridiza-tion stations

HS 400 Pro™ and HS 4800 Pro™ furtherenhance quality page 9

Application BiopharmaNew developments for automation inproteomic researchA robust sample preparation protocol forMALDI-TOF MS analysis of tryptic peptideextracts using the Tecan TeMO-96pipetting robot page 10

DetectionGENios ProTM – the multi-functionalreader proves a hit for multiplexingTecan’s multi-functional microplate readeris used to multiplex HeLa cell-basedviability and apoptosis assays page 12

“Tecan’s good customer service is veryimportant to us”A long-standing customer backs Tecan’scontinuing commitment to customerservice page 14

EventsCalendar Come and meet Tecan atconferences and meetings around theworld in 2005page 16

The Tecan OEM components division inCalifornia’s Silicon Valley has beenmanufacturing OEM instrumentcomponents under the Cavro® brand formore than 30 years. We work directlywith other companies, giving themaccess to our products and collaboratingwith R&D, sales and service organizationsto develop and commercialize validated,automated solutions.

Many leading laboratory instrumentmanufacturers already place their trust inTecan’s Cavro OEM components for thecritical functions of precision liquidhandling and robotics and, as well asrelying on our product performance andreliability, they also benefit from Tecan’sISO 13485 quality system and QSRcompliance.

3CO N T E N T S

The RSP 9000 is an XYZ robotic module.It is the perfect base unit to automate OEM liquid handling applications



4 D E T E C T I O N

Taking microarrays by storm

LS Reloaded & Connect for microarraybatch scanning

Developed by the Custom MicroarrayLaboratory at Novartis, EvanescentResonator (ER) technology produces asignal from fluorescence-basedmicroarray analyses that is up to ahundred times greater than that fromtraditional, passive glass slides. This meansthat less sample material is required togive a strong signal, and there is a bettersignal-to-noise ratio so that evensamples with extremely smallconcentrations can be analyzed. Thesecret of this heightened sensitivity liesin the optical configuration of themicroarray platform, the NovaChip.The surface area built into thesubmicrometer region produces aninterference effect for incident lightand has the effect of inducing stronger resonance. At the same time,the excitation energy is restricted to theultra-thin dielectric layer and this is the

principle behind the evanescentresonator. The restriction of the (laser)energy to the thin surface layergenerates enormous electromagneticforce. These so-called evanescent fieldsstrengthen the fluorescence signal ofmolecules bonded to the surface andcreate a clear demarcation between thatarea and the background fluorescence.

However, to take advantage of thefluorescence strengthening effect of theER principle, it is necessary to adjust theangle of incidence of the excitation beam.The instruments in the LS™ Laser Scannerseries from Tecan are the only commercialmicroarray scanners available that offerthis option and, just as importantly, givehighly reproducible results withmaximum precision*. The LS 200™ hasbeen in daily use at Novartis since 2002and was chosen, not only for the

adjustable angle, but also because of thevariety of measuring modes it offers.

A particular advantage of the newtechnology is the ability to check theapplication of the probe sample, whetherDNA, oligonucleotides or proteins etc.,to the slides. Even if the applicationprocedure is of the highest quality, withsuch a large number of spots there willinevitably be some left without probesample and it is important to registerthese positions before the actualexperiment in order to avoid falsenegative results. With the LS 200 inscatter mode, the buffer residues whichform on each spot when the probe fluidhas been successfully applied can bedetected as salt crystals withoutcompromising the array. The LS 200 isalso completely compatible with a closedstacking device, Connect™ and so thischecking stage can easily be carried outovernight, processing batches of up to200 slides, four at a time.

To take full advantage of the NovaChiptechnology, the quality andreproducibility of hybridization is alsocrucial and for this the Novartis teamalso turned to Tecan. Tecan’s HS 4800™Hybridization Station is completely

A recent collaboration between Tecan andNovartis Pharma AG in Basel has produced a newhighly sensitive microarray technology that hasthe potential to open up new applications andcapabilities for the pharmaceutical industry.

automatic, from pre-hybridization todrying the slides and, as there is nomanual intervention of any sort, it ispractically impossible for the array tobecome contaminated. The small samplevolumes and active mixing of the sampleduring hybridization also help to achievemaximum sensitivity. There is currentlyno comparable hybridization instrumentwhich offers all these features with theadditional benefit of flexibility ofthroughput for up to 48 slides per run.

For the Novartis program, the HS 4800 is set up in the afternoon with up to 24 microarray slides loaded with thefluorescence-marked samples and left torun overnight. Integrated automateddrying of slides with nitrogen protectsfluorescence labels from oxidization untilthey are measured next morning using LS 200 with Connect stacker.

5

More spots and genes are detectable by ER scanning

Signal to background ratio on NovaChip depends on excitation angle

HS 4800 for 48 slides processing

The data are then automatically analyzedusing Array-Pro® Analyzer Software fromMedia Cybernetics based in Silver Spring,MD, USA. This software takes away anychance of operator variability by meansof the patented fixing of the spotlocalization to the exact pixel andautomation of the computer algorithms.

The calculation is carried out withinseconds and is therefore ideally suited to high throughput, fully automatedmethods such as this.

The new technology has proven to beextremely flexible and can be used forany kind of single or dual color arrayapplication, with standard 3 x 1 inchmicroarray glass slides as well as otherglass dimensions and chip formats. Insummary, the NovaChip provides anexcellent solution for extending theapplications of microarray technologyand, at the same time, reduces thenumber of steps in sample preparation.

*The LS Reloaded™, available since late 2004, hasthree times the sensitivity at 532 nm excitationwavelength and twice the sensitivity at 633 nmexcitation wavelength of the LS 200 used by Novartis.

D E T E C T I O N

6 A P P L I C AT I O N B I O P H A R M A


CellerityTM meets customer needs for automated cell culture

Delegates visiting the Tecan stand andguests at a specially held seminar werethrilled to see Cellerity in action and allsigns show that it promises to be themost affordable, compact and flexibleinstrument on the market for cell cultureand maintenance. The new system canbe relied upon to automate all standardcell culture processes such as passaging,splitting, counting and plating. It cansimultaneously grow different cell linesand maintain them in parallel.

Cellerity is the latest product to resultfrom Tecan working together with

partner companies who are leaders in their own particular fields. In thedevelopment of Cellerity and itscorresponding consumables, Tecan,Corning® and Invitrogen GIBCO® havedirectly responded to the needs oflaboratories faced with the constantdemand for a reliable, timely andconsistent supply of high-quality cells.

Cellerity is based on the Freedom EVO®200 platform with an eight-tip liquidhandling arm (LiHa) which has fourchannels for handling cells and four foradding sterile liquids such as media,

additives or buffers. An integrated roboticmanipulator (RoMa) arm transportsplates and flasks around the workspace.The RoMa directly accesses all pipettingstations, incubators, storage devices andother modules. The system can handleautomation-friendly cell culture flasks. Allpipetting and cell manipulation steps areperformed within a laminar flow sterilehood or optionally a class II biosafetycabinet. The robotic incubator controlsCO2, temperature and relative humidityand can hold up to 1000 of both flasksand assay plates. CellGEM™, the newCellerity control software for Cell Growth,

The launch ofCellerityTM, Tecan’sdedicated systemfor automated cellculture, was metwith resoundingenthusiasm at theLab Automation2005 show in SanJosé, California, thispast February.

The pierceable cap of the Corning RoboFlask™ vessel helps maintain a sealed environment duringmedia exchange. It can be removed for manual access.


Tecan’s fully automated system for cellline growth and maintenance will greatlybenefit cell culture laboratories. Thesystem ensures improved quality of cellsfor a wide range of applications and freesup laboratory personnel for otherresearch activities.

7A P P L I C AT I O N B I O P H A R M A

Expansion and Maintenance, is anintuitive software interface that guidesusers through the process of initiating acell line expansion request. A calendar-style interface allows easy selection ofthe dates upon which cells are requiredfor delivery. The CellGEM schedulingsoftware calculates the optimal times forsplitting, harvesting, and plating andthen instructs the instrument to performthose actions automatically. The softwaremaintains a record of the cells grown inthe system, including growth rates andpassage number. Additionally, CellGEMkeeps track of current volumes of mediaand consumables available in the systemand warns users of depleting resources.The whole system can even be leftunattended over a weekend withinstructions to produce cells ready forMonday morning, effectively providingthe laboratory with an extra two workingdays. Bar coding of the flasks means thatthe system knows exactly which protocolto apply to each flask.

Standard T-flasks for cell culture are not automation-friendly and requireexpensive industrial equipment tomanipulate them between stages forstorage, decapping and incubating. Toaddress this problem, Tecan has workedclosely with the Life Sciences Division ofCorning Inc. to develop the automation-friendly RoboFlask™ vessel for Cellerity,designed to meet standard microplatedimensions. These flasks can also behandled by other existing Tecan hardwareand can be stored in instrumentsdesigned to hold microplates, such asincubators, stackers and carousels.

The RoboFlask™ vessel has been validatedfor the successful growth of manydifferent common and rare cell lines, ofhuman and other mammalian origin.With a smaller footprint than a regular75 cm2 flask, the RoboFlask™ vessel has acell growth surface area of 92.6 cm2,allowing the growth of approximately107 cells per flask. The flask cap contains aseptum that can be pierced by the LiHanumerous times without compromising

the flask’s sterility because the cap issterilized with ethanol prior to eachpiercing and otherwise remains closed.Each flask is fitted with a vent with alarge hydrophobic gas permeablemembrane for aeration and pressureexchange.

A high speed AutoLoader™ stores emptyRoboFlask vessels and transfers themdirectly onto a device designed for accessby liquid handling tips, and for shakingand knocking of flasks to harvest cells.Harvested cells may be pumped intoCorning’s ProCulture® spinner flasks,where cell suspensions are diluted asnecessary for plating or seeding of newflasks, based on the measurement of anInnovatis Cedex cell counter. Cellerity hasbeen designed to accommodate GIBCOcell culture media and reagents providingCellerity customers with high-qualitycell culture products efficiently andaffordably. A large refrigerator unit onCellerity holds up to eight bulk GIBCOmedia bags whose contents can bewarmed by an on-line heater to theappropriate temperature immediatelybefore dispensing. The connectionbetween Cellerity’s liquid handlingsystem and GIBCO disposable media bags has been designed to minimize therisks of contamination. Liquids such ascell dissociation products (eg, Trypsin,TrypLE™ Express) or transfection reagents(eg, Lipofectamine™) which are used insmaller volumes can be kept in cooled orheated containers on the Freedom EVOdeck, where they can be directly accessedby the LiHa.

Gentle shaking of cell culture flasks

The robot-friendly flasks are compatible withmany Tecan automation options including theRoMa arm on the Freedom EVO and theAutoLoader

8 D E T E C T I O N /A P P L I C AT I O N B I O P H A R M A


Hereditary non-polyposis colorectal cancer(HNPCC) is a familial syndrome that gives amuch higher risk of developing coloncancer and accounts for 2-5 % of all cases.Two different molecular pathways arethought to be involved in the developmentof this disease, one being punctualalterations of the DNA, and the other,chromosomal instabilities inmicrosatellites. Dr Wolfgang Dietmaier andcolleagues at the Regensburg UniversityClinic in Germany have been using Tecan’sHS 400 Hybridization Station to comparegenome-wide DNA profiles in samplesfrom these two variants. It has alreadybeen shown that microsatellite-stable(MSS) and microsatellite-instable (MSI)tumor variants can be histologicallydifferentiated and the aim of this studywas to compare the frequency distributionof individual genes in tumor tissue withhealthy tissue.

DNA was extracted from surgicallyremoved tumor and normal mucosasamples and labelled with Cy3® and Cy5®red fluorescence, respectively. It was thenhybridized against arrays of a defined BAClibrary of healthy samples, consisting of 32blocks (4 x 8 blocks), each approximately640 spots, dried on slides. This alloweddirect comparison of the DNA copynumbers of the pathological and healthysamples, where DNA gains in a definedlocus in the tumor DNA were representedby red spots, and DNA losses wererepresented by green spots. If the tumorDNA was present in equal amounts to thatin the normal mucosa (i.e. a healthy ratio),then the two labels neutralized each otherto an orange colour.

The HS 400 Hybridization Station madethe procedure extremely simple. The arrays

HS 400TM Hybridization Station makeslight work of genomic DNA patterns

were placed in the special slide holder andthe carrier was then inserted into theinstrument in dry form. The entire methodwas carried out in stages – pre-washing,insertion of the hybridization sample,hybridization, four washing steps and,finally, drying with nitrogen – and theindividual times, temperatures and buffersfor each stage were optimized for thisparticular application.

This application highlighted a number ofadvantages that the HS 400 has over other

hybridization stations, many of whichconcentrate on minimizing the risk ofcontamination and other artifacts atdifferent stages throughout hybridization.The entirely automated process takes placein the unique four microarray slide holderincluding the final drying step, whichmeans that the arrays are dry when theyenter and leave the instrument and do notneed to be handled throughout thehybridization.

Cy3 and Cy5 are registered trademarks of Amersham, Inc.

Figure 1: Example of one block of themicroarray used for profiling ofhereditary non-polyposis colorectalcancer (HNPCC). Automatedprocessing in Tecan’s HS 400 leads tofewer background artifacts, highersensitivity and specificity comparedto other procedures.

9D E T E C T I O N /A P P L I C AT I O N B I O P H A R M A


Tecan’s HS Series of hybridization stationsare well proven tools to automate avariety of microarray applications, includ-ing DNA and protein arrays, and in situhybridization.

The new HS 400 Pro and HS 4800 Procontain Tecan’s unique ABS™ (Active BubbleSuppression) system, which preventsformation of bubbles within the chambers,eliminating hybridization artifacts andenhancing consistency of results. Noveldual chambers are easily interchangeablewith single area chambers and enablethe new systems to fully automate theprocessing of two independent sub-arrays per slide with no risk of carry-over.

HS 400 ProTM and HS 4800 ProTM

share the benefits of Tecan’sexisting hybridization systems:

• Fully automated hybridization – frompre-hybridization to automated slidedrying

• Less background, fewer artifacts,higher sensitivity

• Designed to maximize reproducibilityand reliability of results

• Easy to use, low maintenance • Low volume hybridization chambers

save precious samples• Two models can handle a variety of

throughputs

Tecan prepares launch ofnew hybridization stations

HS 400 Pro™ and HS 4800 Pro™ furtherenhance quality, repro-ducibility and applicationflexibility for microarrayhybridizations

D E T E C T I O N /A P P L I C AT I O N B I O P H A R M A

New products

Matrix-Assisted Laser Desorption IonizationTime-of-Flight (MALDI-TOF) mass spectro-metry (MS) is a key technique for theanalysis of proteins and peptides in pro-teomic research. It can be used to analyzemolecules over a wide molecular weightrange from a few hundred daltons up to300kDa with very high accuracy and withdetection sensitivities in the low fmolrange. It is very useful in peptide massfingerprint analysis of large proteins.

Sample preparation and the preparationof an appropriate analyte/matrix mixtureare critical limiting factors in MALDI-TOFmass spectrometric analysis. To achievehigh sensitivity, impurities such as saltsand buffer components must be washedaway efficiently with minimal loss ofanalytes.

Figure 1: MALDI sample preparationperformed on a Tecan Genesis Workstationwith a TeMO-96 multi-pipetting unit

10 A P P L I C AT I O N B I O P H A R M A


The two experiments described belowevaluate a robust sample preparationprotocol for MALDI-TOF MS analysis oftryptic peptide extracts using the TecanTeMO-96 pipetting robot (Figure 1), whichaims to make MALDI-TOF more accessiblefor high throughput applications.

Method

Our sample preparation is based on the α-cyano-4-hydroxycinnamic acid (CHCA)affinity preparation method (Gobom etal. (2001), Anal. Chem. 73, 434-438) inwhich the tryptic peptide extracts areapplied onto a pre-structured anchorMALDI sample plate (Scout-MTP 384/600AnchorChip; Bruker Daltonics, Bremen,Germany) prepared with a thin layer ofthe MALDI matrix CHCA. The TeMOworktable accommodates eight 96-wellmicrotiter plates with peptide samplesand two MALDI sample plates, each with384 sample positions. The TeMO-96pipetting head transfers 1 µL of eachpeptide sample from the microtiterplates to the MALDI sample plate wherethe droplets are allowed to dry. New racksof disposable pipette tips are delivered bythe Tecan TeStack for each set of 96tryptic extracts to avoid sample crosscontamination. After the tryptic digestsamples have dried, contaminants arewashed away by dispensing 3 µL dropletsof 0.1 % trifluoroacetic acid (TFA) onto thesample, followed by aspiration of thedroplet after three seconds. Betweenwashing of different sample spots, thepipette tips were rinsed using the Tecanwashing station in a multi-stepprocedure included in the MALDI samplewashing procedure. After emptying thetips, two cleaner steps with 90 µL MilliQwater each were employed, with soak

times of 1 second and flow through set at80. The first step included three cycles,with change of 5 µL each, and the secondstep included two cycles without change.

Controlling carry-over during MALDIsample plate preparation

The consumption of pipette tips duringwashing of sample spots on the MALDIsample plates can be minimized by usingthe same set of tips for washing of allsamples on an entire sample plate, aslong as the pipette tips can be rinsedsufficiently between washing of differentsample spots on the MALDI sample plate.The efficiency of the pipette tip washingprocedure was tested by preparing a setof three single peptides on adjacentsample spots on the MALDI sample plate(Figure 2), followed by washing the wholesample plate with one set of pipette tips.Angiotensin I, neurotensin and ACTH 18-39 were added onto quadrant Q1, Q2 andQ3 respectively. Nothing was added toquadrant Q4. The four quadrants werewashed using the previously mentionedprotocol with two cycles of 3 µL of 0.1 %TFA each. As shown in the MALDI-TOFmass spectra in Figure 2, no carry-over ofthe peptide prepared on the previoussample spot could be detected on thefollowing spot. None of the peptidesprepared on quadrants Q1 to Q3 weredetected in the mass spectrum fromquadrant Q4. The preparation of thethree single peptides was performed in 32parallel sets, with no carry-over detectedin any of the mass spectra.

Assessing reproducibility ofpreparation for MALDI-TOF MS

The reproducibility of the automaticsample application and MALDI sample

Dr. Niklas Gustavsson*, Dr. Dieter Weichart,Dr. Johan Gobom, Max Planck Institute forMolecular Genetics, Berlin

*now based at the Mass Spectrometry group,Dept of Plant Biochemistry, Lund University, Sweden

New developments for automationin proteomic research:MALDI sample preparationfor high throughput peptide mass fingerprinting

Dr. Niklas Gustavsson, in his lab at the MPI for Molecular Genetics


11A P P L I C AT I O N B I O P H A R M A

plate washing procedures was tested bypreparing the same set of 368 trypticdigests of human proteins excised from atwo-dimensional gel onto two MALDIsample plates. The plates were processedin parallel, and spectra were acquiredunder identical conditions on an UltraflexLIFT mass spectrometer (Bruker Daltonics,Bremen, Germany). Using the MASCOTsoftware, the peptide mass fingerprintdata were submitted to a databasesearch in the Swiss protein database(http://www.expasy.org/sprot/),considering scores larger than 62indicative of reliable identification(p<0.005). With these settings, 98proteins could be identified in total, ofwhich 78 (79.6 %) were identified fromboth MALDI sample plates (see Figure 3a).Three samples appeared to yieldcontradicting identifications in the twopreparations. Upon closer inspection,however, the spectra were found tocontain peptide signals from twodifferent proteins. In each sample, bothproteins were identified, but withdifferent rank. Hence no conflictingresults were observed between the twoparallel MALDI sample plate preparations,indicating high reproducibility of bothrobotic handling and mass spectrometricidentification. For demonstration, Figure3b shows a selection of representativepairs of spectra originating from the twoMALDI sample plates.

Summary

In conclusion, the results presented here show that MALDI sample platepreparation using the Tecan TeMO unitallows robust, reproducible, simple andhighly parallel analysis of protein digests.They also demonstrate that the TeMOwashing station saves pipette tips duringthe MALDI sample plate washingprocedure, and that TeMO dispensessmall volumes of protein digests withsufficient reliability to enable highthroughput application of MALDI-MSbased peptide mass fingerprinting.

Figure 2: MALDI-TOF mass spectra analysis to test the efficiency of the pipette tip washing procedure

Figure 3 (a): Overlap between proteinidentification results from two parallel MALDIruns with target plates supplied with identicalsamples using the Tecan TeMO module: Proteinsidentified on the first plate symbolized by redcolour, and those identified from the secondplate by blue colour. The number of proteinsidentified is shown below. The overlap from atotal of 98 identified proteins was 79.6 %,without any conflicts of identification.

(b): Examples of corresponding sections of massspectra obtained for identical samples on thetwo MALDI targets (first target: red spectra,second target: blue spectra)

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12 D E T E C T I O N


GENios ProTM

– the multi-functional readerproves a hit for multiplexing

However, for multiplexing to really work,there is the challenge of developinghomogenous assays that do not requirewashing and sample handling steps,and the requirement of excellentperformance, flexibility and multi-functionality from the detectioninstrument used.

Prof. Dr. Gadek-Wesierski and her researchteam at the Medical University of Viennahave shown that the GENios Pro multi-functional microplate reader from Tecanmeets all these criteria and has a numberof features such as individual optics for all reading modes that make itparticularly suitable for multiplexing ofcell-based assays with various readouts .

The team in Vienna aimed to multiplextwo cell-based assays having fluorescent

and luminescent output to obtaininformation regarding viability andapoptosis directly from the same samplewell, using Cisplatin (CP) as a testsubstance for treating HeLa cells. CP is awidespread anticancer drug used forchemotherapy. It inhibits viability andproliferation and induces apoptosis inhuman cervical carcinoma cells (seeFigure 1).

In the experiment, HeLa cells S3 werecultured in 96-well plates and treatedwith increasing concentrations of CP, theCellTiter-Blue® Cell Viability Assay fromPromega was used to calculate viability.This assay determines the number ofmetabolically active cells based on thedirect addition of resazurin to cells in aculture medium, which is then convertedinto resorufin by the cells. Measurements

Multiplexing assays for any applications offer a range of benefits overstandard assays. In addition to being amenable to high throughput, theyalso allow the measurement and analysis of more than one parametersimultaneously and save time and money by conserving precious compoundsand cell culture reagents.

Dr. Gerlinde Zerza-Schnitzhofer,Dr. Manfred Lansing, Mag. MarietaGueorguieva1, Mag. Carmen Ranftlerand A.o.Univ. Prof. Dr. Jozefa Gadek-Wesierski1

Tecan Austria GmbH, Grödig, Austria and1Medical University of Vienna, Vienna, Austria

Figure 1: Detection of apoptotic HeLa cells.Untreated HeLa cells and HeLa cells treated with40 µM cisplatin (15 hours) were fixed withmethanol and stained with a fluorescein-conjugated monoclonal mouse antibody (M30-CytoDeath, Roche, Vienna, Austria). TheCytoDeath antibody is specifically for caspase-3cleaved cytokeratin-18 and stains cells in variousstages of apoptosis. Cell nuclei were madevisible using DAPI. Mitotic (M) and apoptotic (A)cells are marked with the following symbol ->.

13C U STO M E R S U P P O RT


were taken after the appropriateincubation period using the GENios Proreader (Figure 2). Resorufin is activated at545 nm and emission is detected at590 nm. The fluorescence reading canthen be correlated with the number ofmetabolically active, living cells, ifappropriate serial dilutions of cells havebeen performed accordingly.

In experiments with adherent cells,excitation and detection through thebottom of a microplate are preferable totop reading as this reduces disruptiveinfluences of the culture medium on themeasurement signal. A preliminary testwith CellTiter-Blue® showed that thedetection limits in bottom reading modewere between six and eight times lowerthan in top reading mode. This meansthat fewer cells and materials can beused to achieve the same results, which isparticularly advantageous for cell-basedapplications.

the sample wells containing the CellTiter-Blue reagents, after having taken theviability readings. The substrate solutioncontains a stabilized luciferase and isoptimized for caspase/luciferase activityand cell lysis. The substrate solutiondestroys the cells, the preluminescentsubstrate is cleaved by active caspasesreleasing luciferin that is then consumedby the luciferase. The resultingluminescent signal is proportional tocaspase activity and can be measured.The results in Figure 3 show a clearincrease in apoptosis at high CPconcentrations. The number of apoptoticcells increased in this experiment byapproximately four fold. Correlating thereadings from both the viability andapoptosis analyses with the number ofcells allows the percentage of apoptoticcells to be determined.

Summary

Multiplexing is an efficient way ofobtaining more than one set ofexperimental data from the same samplewell in order to better understand

complex cellular processes. Additionally,multiplexing offers the advantage ofreducing experimental variation, likefluctuations in cell numbers due to cellclumping and dispensing. Homogenousassays, which require no washing andseparation stages, can easily beintegrated into Tecan Robotic systemswith the GENios Pro Reader for use inhigh throughput screening. Stages suchas the addition of various assay reagentsby injectors into 96 and 384 well plates,incubation at 37° C and plate-shaking,can all be carried out in this modularreader. The measurement methodsavailable on the GENios Pro are suitablefor a large number of multiplex assaysand range from absorption andfluorescence (FI, FRET, TRF) to variousluminescence techniques (BRET2™, Glowand Flash Type Luminescence).

This experiment combined two cell-basedassays in one multiplex experiment but,using additional assays, e.g. LDH releaseor reporter gene assays, the teambelieves that multiplexing could easily beextended to answer further questions.

Acknowledgement

We would like to thank Prof. Dr. Gadek-Wesierski and her team at the Universityof Vienna for providing the data andDr. Katarina Bohm and Dr. Heinz Winklerfrom Promega for kindly providing thereagents.GENios Pro™ is a trademark of Tecan Group Ltd.Cell Titer-Blue® and Caspase-Glo 3/7™ Assays are trademarksof Promega corporation.BRET2™ is a proprietary technology from BioSignal Packard.Patents pending.

Prof. Dr. Gadek-Wesierski and herresearch team are based at theInstitute of Cancer Research at theMedical University of Vienna. Theinstitute is focused on basic cancerresearch, scientific education and thedevelopment of a new understandingof cancer disease.

The group is studying the mechanismsof cell cycle regulation duringmalignant transformation of cells. Theexpression, activation and inhibition oftumor suppression proteins (eg, p53),

Figure 2: GENios Pro microplate reader. Featuressuch as rapid fluorescence bottom reading andreagent addition by injectors make thismultimode reader particularly suitable for alarge number of cell-based assays in HTS.

Figure 3: Multiplex analysis of viability andapoptosis in HeLa cells. Viability was analyzedusing CellTiter-Blue® Assay and apoptosis usingCaspase-Glo 3/7™ Assay following incubation ofthe cells in 96-well microplates with 0 – 40 µMcisplatin (CP).

Figure 3 shows the results of the CellTiter-Blue® assay after the HeLa cells wereincubated for 24 hours with variousconcentrations of CP (0, 1, 5, 20, 40 µM).As the CP concentration increases, thenumber of metabolically active cells isreduced by approximately 70 %.Information on the apoptosis rate wasobtained in the multiplex assay using thePromega Caspase-Glo 3/7™ assay tomeasure the activity of caspase-3 and -7,both of which play a key role in theapoptosis of mammalian cells. Caspaseactivity is measured by adding apreluminescent substrate solution into

which play a key role in cycle control, areinvestigated in different cell lines andspecies. Additionally, the group is studyingthe effect of new anti-cancer drugs, inparticular regarding their potential forreactivating the tumor suppressor proteinp53, which results in enhanced efficiencyof chemotherapy treatment.

http://www.p53parp.at

D E T E C T I O N

14 C U STO M E R S U P P O RT

Just afriendlyphonecall away

Tecan Journal 2005


15C U STO M E R S U P P O RT

What is the main role of your researchfacilities?

As a whole, the Crop Protection ResearchFacilities are responsible for investigatingchemicals that have the potential toprotect crops, mainly fungicides,insecticides and herbicides. The site inBasel is essentially a dispensary, where allthe research compounds are stored,prepared into solution and pipetted byTecan systems into reaction microplates.These plates are then transferred to thefacility in Stein where we perform arange of preliminary and validatory tests,also using Tecan systems, on thechemicals themselves, and on the effectsthey have on plants in differentformulations and concentrations.

What level of sample throughput are youachieving?

At the moment our throughput is around200-300 compounds per week, not whatI would call high throughput at all, butwe are performing numerous tests atmany different concentrations with eachindividual compound.

What Tecan equipment do you have andhow long have you been using it?

We have been using Tecan equipmentsince we started operations seven yearsago. At the Basel site, we have twoGENESIS RSPs with liquid handling (LiHa)

arms on each for pipetting. A FreedomEVO and carousel handle the plates sothe process is completely automated. InStein we have GENESIS RSPs equippedwith LiHa and robotic manipulator(RoMa) arms, carousels and speciallyadapted needles that spray thechemicals onto the plants.

Did Tecan provide you with applicationsupport to set up these processes?

Yes, we have had application supportfrom the beginning. At first, ourequipment was set up for highthroughput screening but every time wehave a new project or change the processwe have needed to adapt the equipment.Sometimes we can make the changesourselves, but if we need instrumentupgrades or other alterations we alwaysdirectly involve Tecan.

And how do you rate Tecan’s customerservice?

Very good! First and foremost, theequipment is generally very reliable.However, whenever there is a problem, theTecan engineers guide us through somegeneral checks so that they can fullyanalyse the problem and advise whetherthere is some way we can resolve theproblem ourselves. If not, they are with uswithin 24 hours or if possible, because weare quite close, within 3 hours. Tecan’s goodcustomer service is very important to us.

Tecan CustomerSupportWhether the customer needsassistance in reliable automation,precise and safe liquid handling, ormaintenance and operation of roboticsand detection products, services arein place to assist the customer at alllevels of activity. In this way, Tecan isable to offer the expertise necessaryto ensure professional use and high-performance operation ofTecan equipment.

Laurent Nussbaumer is Supporting Engineer atSyngenta’s Crop Protection Research facilitiesbased in Basel and Stein in Switzerland.Mr Nussbaumer is responsible for equipment atboth sites and has worked with Tecan on severalprojects over a period of seven years.

Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.comPhotography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.netEditor: kdm/UK www.kdm-communications.comPrint: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Switzerland AG, Marketing Communication, Seestrasse 103, CH-8708 Männedorf, [email protected], www.tecan.com

©2005, Tecan Trading AG, Switzerland, all rights reserved


Tecan Journal 1/2005 www.tecan.com

Conferences and trade shows 2005 preview

Europe

SBS Geneva September 11-15 2005

Biotech Forum and Scanlab Stockholm October 10-12 2005

Biotechnica Hannover October 18-20 2005

JIB – Journées Internationales de Biologie Paris November 3-5 2005

International Biotech and Lab Automation Europe London November 15-16 2005

Medica Düsseldorf November 16-19 2005

Expoquimia Barcelona November 14-18 2005

The 5th Protein Science Society of Japan Annual Meeting Fukuoka June 30-July 2 2005

Japan

The Japan Society for Clinical Laboratory Automation Yokohama September 28-30 2005

The 28th Annual Meeting of Molecular Biology Society of Japan Fukuoka December 7-10 2005

Drug Discovery Boston, MA August 7-12 2005

AACC Orlando, FL July 24-28 2005

USA

Chips to Hits Boston, MA September 12-15 2005

International Symposium on Human Identification (Promega) Grapevine, Texas September 26-29 2005

Neuroscience Washington, DC November 12-16 2005

ASHG Salt Lake City, Utah October 26-29 2005

AABB Seattle, WA October 16-18 2005

Cell Biology (ASCB) San Francisco December 10-14 2005

Australia

2. ComBIO 2005 Adelaide September 25-29 2005

Asian Pacific Congress of Clinical Biochemistry Brisbane September 14-18 2005

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Documents

Tecan Journal Edition 01/2005