Tecan Journal Edition 01/2005

ISSN 1660-5276

Tecan Journal Edition 1 2005

Cellerity™ meets customer needs for automated cell culture page 6

HS 400™ Hybridization Station makes light work of genomic DNA patterns page 8

Talk to Tecan’s Customer Support page 14

2

GLOBAL N EWS

Welcome to the “new look” Tecan Journal Firstly, we would like to take this opportunity to introduce Thomas Bachmann, who joined Tecan in February 2005 as the new CEO. Thomas brings a wealth of experience in international senior management to his new role and will be focusing his efforts on employees, customers and investors alike.

“My strong personal commitment to Tecan is to continue developing excellent relationships with our customers and Thomas Bachmann Chief Executive Officer partners, and to maintain (CEO) their confidence and trust. We will focus on issues that are important to our customers and aim to win the respect of the life science, diagnostic and pharmaceutical worlds by consistently providing the solutions and support they need.”

Our presence in the Asia-Pacific region has been boosted by the opening of the Tecan office in Beijing. Our new Tecan representatives are Mark Wang (mark.wang@tecan.com) and Barbara Stobbe (barbara.stobbe@tecan.com).

Distributors in this region recently met at the Asia Pacific Distributor Sales Meeting 2005 in Bangkok. The meeting was hosted by Marco Vittoz, Director Sales International, and attended by more than 20 members of Tecan South East Asia (SEA) distributors and Tecan representatives from China and (SEA), as well as new area managers for India and New Zealand. During the meeting, information was shared between Tecan and the distributors on a personal basis and Tecan’s sales and support strategy in the Asia Pacific market was discussed.

Asia Pacific Distributor Sales Meeting 2005 in Bangkok

Barbara Stobbe and Mark Wang

Tecan Journal 1/2005

Training Mark Wang at Tecan Switzerland and Austria

CO N T E N T S

The Tecan OEM components division in California’s Silicon Valley has been manufacturing OEM instrument components under the Cavro® brand for more than 30 years. We work directly with other companies, giving them access to our products and collaborating with R&D, sales and service organizations to develop and commercialize validated, automated solutions.

Many leading laboratory instrument manufacturers already place their trust in Tecan’s Cavro OEM components for the critical functions of precision liquid handling and robotics and, as well as relying on our product performance and reliability, they also benefit from Tecan’s ISO 13485 quality system and QSR compliance.

Detection

Application Biopharma

Taking microarrays by storm Tecan teams up with Novartis to create an impressive new microarray technology with the potential for use in novel applications page 4

New developments for automation in proteomic research A robust sample preparation protocol for MALDI-TOF MS analysis of tryptic peptide extracts using the Tecan TeMO-96 pipetting robot page 10

Application Biopharma CellerityTM meets customer needs for automated cell culture Cellerity, Tecan’s new fully automated system for cell line growth and maintenance, promises to be the most affordable, compact and flexible instrument of its kind page 6

Detection/Application Biopharma

Detection GENios ProTM – the multi-functional reader proves a hit for multiplexing Tecan’s multi-functional microplate reader is used to multiplex HeLa cell-based viability and apoptosis assays page 12 “Tecan’s good customer service is very important to us” A long-standing customer backs Tecan’s continuing commitment to customer service page 14

HS 400TM Hybridization Station makes light work of genomic DNA patterns The HS 400 is used to investigate molecular changes along different pathways in hereditary non-polyposis colorectal cancer page 8 Tecan prepares launch of new hybridization stations The RSP 9000 is an XYZ robotic module.

It is the perfect base unit to automate OEM liquid handling applications

HS 400 Pro™ and HS 4800 Pro™ further enhance quality page 9

Events Calendar Come and meet Tecan at conferences and meetings around the world in 2005 page 16

Tecan Journal 1/2005

3

4

G EL O D T EBCATLI O NN EWS

Taking microarrays by storm

LS Reloaded & Connect for microarray batch scanning

A recent collaboration between Tecan and Novartis Pharma AG in Basel has produced a new highly sensitive microarray technology that has the potential to open up new applications and capabilities for the pharmaceutical industry.

Developed by the Custom Microarray Laboratory at Novartis, Evanescent Resonator (ER) technology produces a signal from fluorescence-based microarray analyses that is up to a hundred times greater than that from traditional, passive glass slides. This means that less sample material is required to give a strong signal, and there is a better signal-to-noise ratio so that even samples with extremely small concentrations can be analyzed. The secret of this heightened sensitivity lies in the optical configuration of the microarray platform, the NovaChip.

The surface area built into the submicrometer region produces an interference effect for incident light and has the effect of inducing stronger resonance. At the same time, the excitation energy is restricted to the ultra-thin dielectric layer and this is the Tecan Journal 1/2005

principle behind the evanescent resonator. The restriction of the (laser) energy to the thin surface layer generates enormous electromagnetic force. These so-called evanescent fields strengthen the fluorescence signal of molecules bonded to the surface and create a clear demarcation between that area and the background fluorescence.

However, to take advantage of the fluorescence strengthening effect of the ER principle, it is necessary to adjust the angle of incidence of the excitation beam.

The instruments in the LS™ Laser Scanner series from Tecan are the only commercial microarray scanners available that offer this option and, just as importantly, give highly reproducible results with maximum precision*. The LS 200™ has been in daily use at Novartis since 2002 and was chosen, not only for the

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

A particular advantage of the new technology is the ability to check the application of the probe sample, whether DNA, oligonucleotides or proteins etc., to the slides. Even if the application procedure is of the highest quality, with such a large number of spots there will inevitably be some left without probe sample and it is important to register these positions before the actual experiment in order to avoid false negative results. With the LS 200 in scatter mode, the buffer residues which form on each spot when the probe fluid has been successfully applied can be detected as salt crystals without compromising the array. The LS 200 is also completely compatible with a closed stacking device, Connect™ and so this checking stage can easily be carried out overnight, processing batches of up to 200 slides, four at a time.

To take full advantage of the NovaChip technology, the quality and reproducibility of hybridization is also crucial and for this the Novartis team also turned to Tecan. Tecan’s HS 4800™ Hybridization Station is completely

DETECTION

The calculation is carried out within seconds and is therefore ideally suited to high throughput, fully automated methods such as this.

HS 4800 for 48 slides processing

automatic, from pre-hybridization to drying the slides and, as there is no manual intervention of any sort, it is practically impossible for the array to become contaminated. The small sample volumes and active mixing of the sample during hybridization also help to achieve maximum sensitivity. There is currently no comparable hybridization instrument which offers all these features with the additional benefit of flexibility of throughput for up to 48 slides per run.

The data are then automatically analyzed using Array-ProÂŽ Analyzer Software from Media Cybernetics based in Silver Spring, MD, USA. This software takes away any chance of operator variability by means of the patented fixing of the spot localization to the exact pixel and automation of the computer algorithms.

The new technology has proven to be extremely flexible and can be used for any kind of single or dual color array application, with standard 3 x 1 inch microarray glass slides as well as other glass dimensions and chip formats. In summary, the NovaChip provides an excellent solution for extending the applications of microarray technology and, at the same time, reduces the number of steps in sample preparation. *The LS Reloaded™, available since late 2004, has three times the sensitivity at 532 nm excitation wavelength and twice the sensitivity at 633 nm excitation wavelength of the LS 200 used by Novartis.

For the Novartis program, the HS 4800 is set up in the afternoon with up to 24 microarray slides loaded with the fluorescence-marked samples and left to run overnight. Integrated automated drying of slides with nitrogen protects fluorescence labels from oxidization until they are measured next morning using LS 200 with Connect stacker.

More spots and genes are detectable by ER scanning

Signal to background ratio on NovaChip depends on excitation angle

5

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

The launch of CellerityTM, Tecan’s dedicated system for automated cell culture, was met with resounding enthusiasm at the Lab Automation 2005 show in San José, California, this past February.

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

Delegates visiting the Tecan stand and guests at a specially held seminar were thrilled to see Cellerity in action and all signs show that it promises to be the most affordable, compact and flexible instrument on the market for cell culture and maintenance. The new system can be relied upon to automate all standard cell culture processes such as passaging, splitting, counting and plating. It can simultaneously grow different cell lines and maintain them in parallel.

Cellerity is the latest product to result from Tecan working together with

Tecan Journal 1/2005

partner companies who are leaders in their own particular fields. In the development of Cellerity and its corresponding consumables, Tecan, Corning® and Invitrogen GIBCO® have directly responded to the needs of laboratories faced with the constant demand for a reliable, timely and consistent supply of high-quality cells.

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

additives or buffers. An integrated robotic manipulator (RoMa) arm transports plates and flasks around the workspace.

The RoMa directly accesses all pipetting stations, incubators, storage devices and other modules. The system can handle automation-friendly cell culture flasks. All pipetting and cell manipulation steps are performed within a laminar flow sterile hood or optionally a class II biosafety cabinet. The robotic incubator controls CO2, temperature and relative humidity and can hold up to 1000 of both flasks and assay plates. CellGEM™, the new Cellerity control software for Cell Growth,

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

Expansion and Maintenance, is an intuitive software interface that guides users through the process of initiating a cell line expansion request. A calendarstyle interface allows easy selection of the dates upon which cells are required for delivery. The CellGEM scheduling software calculates the optimal times for splitting, harvesting, and plating and then instructs the instrument to perform those actions automatically. The software maintains a record of the cells grown in the system, including growth rates and passage number. Additionally, CellGEM keeps track of current volumes of media and consumables available in the system and warns users of depleting resources.

The whole system can even be left unattended over a weekend with instructions to produce cells ready for Monday morning, effectively providing the laboratory with an extra two working days. Bar coding of the flasks means that the system knows exactly which protocol to apply to each flask.

Standard T-flasks for cell culture are not automation-friendly and require expensive industrial equipment to manipulate them between stages for storage, decapping and incubating. To address this problem, Tecan has worked closely with the Life Sciences Division of Corning Inc. to develop the automationfriendly RoboFlask™ vessel for Cellerity, designed to meet standard microplate dimensions. These flasks can also be handled by other existing Tecan hardware and can be stored in instruments designed to hold microplates, such as incubators, stackers and carousels.

The RoboFlask™ vessel has been validated for the successful growth of many different common and rare cell lines, of human and other mammalian origin.

With a smaller footprint than a regular 75 cm2 flask, the RoboFlask™ vessel has a cell growth surface area of 92.6 cm2, allowing the growth of approximately 107 cells per flask. The flask cap contains a septum that can be pierced by the LiHa numerous times without compromising

the flask’s sterility because the cap is sterilized with ethanol prior to each piercing and otherwise remains closed.

Each flask is fitted with a vent with a large hydrophobic gas permeable membrane for aeration and pressure exchange.

A high speed AutoLoader™ stores empty RoboFlask vessels and transfers them directly onto a device designed for access by liquid handling tips, and for shaking and knocking of flasks to harvest cells.

Harvested cells may be pumped into Corning’s ProCulture® spinner flasks, where cell suspensions are diluted as necessary for plating or seeding of new flasks, based on the measurement of an Innovatis Cedex cell counter. Cellerity has been designed to accommodate GIBCO cell culture media and reagents providing Cellerity customers with high-quality cell culture products efficiently and affordably. A large refrigerator unit on Cellerity holds up to eight bulk GIBCO media bags whose contents can be warmed by an on-line heater to the appropriate temperature immediately before dispensing. The connection between Cellerity’s liquid handling system and GIBCO disposable media bags has been designed to minimize the risks of contamination. Liquids such as cell dissociation products (eg, Trypsin, TrypLE™ Express) or transfection reagents (eg, Lipofectamine™) which are used in smaller volumes can be kept in cooled or heated containers on the Freedom EVO deck, where they can be directly accessed by the LiHa.

Tecan’s fully automated system for cell line growth and maintenance will greatly benefit cell culture laboratories. The system ensures improved quality of cells for a wide range of applications and frees up laboratory personnel for other research activities.

Gentle shaking of cell culture flasks

The robot-friendly flasks are compatible with many Tecan automation options including the RoMa arm on the Freedom EVO and the AutoLoader

Tecan Journal 1/2005

7

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

HS 400TM Hybridization Station makes light work of genomic DNA patterns

Hereditary non-polyposis colorectal cancer (HNPCC) is a familial syndrome that gives a much higher risk of developing colon cancer and accounts for 2-5 % of all cases.

Two different molecular pathways are thought to be involved in the development of this disease, one being punctual alterations of the DNA, and the other, chromosomal instabilities in microsatellites. Dr Wolfgang Dietmaier and colleagues at the Regensburg University Clinic in Germany have been using Tecan’s HS 400 Hybridization Station to compare genome-wide DNA profiles in samples from these two variants. It has already been shown that microsatellite-stable (MSS) and microsatellite-instable (MSI) tumor variants can be histologically differentiated and the aim of this study was to compare the frequency distribution of individual genes in tumor tissue with healthy tissue.

DNA was extracted from surgically removed tumor and normal mucosa samples and labelled with Cy3® and Cy5® red fluorescence, respectively. It was then hybridized against arrays of a defined BAC library of healthy samples, consisting of 32 blocks (4 x 8 blocks), each approximately 640 spots, dried on slides. This allowed direct comparison of the DNA copy numbers of the pathological and healthy samples, where DNA gains in a defined locus in the tumor DNA were represented by red spots, and DNA losses were represented by green spots. If the tumor DNA was present in equal amounts to that in the normal mucosa (i.e. a healthy ratio), then the two labels neutralized each other to an orange colour.

The HS 400 Hybridization Station made the procedure extremely simple. The arrays Tecan Journal 1/2005

were placed in the special slide holder and the carrier was then inserted into the instrument in dry form. The entire method was carried out in stages – pre-washing, insertion of the hybridization sample, hybridization, four washing steps and, finally, drying with nitrogen – and the individual times, temperatures and buffers for each stage were optimized for this particular application.

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

hybridization stations, many of which concentrate on minimizing the risk of contamination and other artifacts at different stages throughout hybridization.

The entirely automated process takes place in the unique four microarray slide holder including the final drying step, which means that the arrays are dry when they enter and leave the instrument and do not need to be handled throughout the hybridization.

Cy3 and Cy5 are registered trademarks of Amersham, Inc.

Figure 1: Example of one block of the microarray used for profiling of hereditary non-polyposis colorectal cancer (HNPCC). Automated processing in Tecan’s HS 400 leads to fewer background artifacts, higher sensitivity and specificity compared to other procedures.

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

Tecan prepares launch of new hybridization stations HS 400 Pro™ and HS 4800 Pro™ further enhance quality, reproducibility and application flexibility for microarray hybridizations Tecan’s HS Series of hybridization stations are well proven tools to automate a variety of microarray applications, including DNA and protein arrays, and in situ hybridization.

The new HS 400 Pro and HS 4800 Pro contain Tecan’s unique ABS™ (Active Bubble Suppression) system, which prevents formation of bubbles within the chambers, eliminating hybridization artifacts and enhancing consistency of results. Novel dual chambers are easily interchangeable with single area chambers and enable the new systems to fully automate the processing of two independent subarrays per slide with no risk of carry-over.

HS 400 ProTM and HS 4800 ProTM share the benefits of Tecan’s existing hybridization systems: • Fully automated hybridization – from pre-hybridization to automated slide drying • Less background, fewer artifacts, higher sensitivity • Designed to maximize reproducibility and reliability of results • Easy to use, low maintenance • Low volume hybridization chambers save precious samples • Two models can handle a variety of throughputs

Tecan Journal 1/2005

9

10

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

New developments for automation in proteomic research: MALDI sample preparation for high throughput peptide mass fingerprinting Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS) is a key technique for the analysis of proteins and peptides in proteomic research. It can be used to analyze molecules over a wide molecular weight range from a few hundred daltons up to 300kDa with very high accuracy and with detection sensitivities in the low fmol range. It is very useful in peptide mass fingerprint analysis of large proteins.

Sample preparation and the preparation of an appropriate analyte/matrix mixture are critical limiting factors in MALDI-TOF mass spectrometric analysis. To achieve high sensitivity, impurities such as salts and buffer components must be washed away efficiently with minimal loss of analytes.

Figure 1: MALDI sample preparation performed on a Tecan Genesis Workstation with a TeMO-96 multi-pipetting unit

Tecan Journal 1/2005

The two experiments described below evaluate a robust sample preparation protocol for MALDI-TOF MS analysis of tryptic peptide extracts using the Tecan TeMO-96 pipetting robot (Figure 1), which aims to make MALDI-TOF more accessible for high throughput applications.

Method Our sample preparation is based on the α-cyano-4-hydroxycinnamic acid (CHCA) affinity preparation method (Gobom et al. (2001), Anal. Chem. 73, 434-438) in which the tryptic peptide extracts are applied onto a pre-structured anchor MALDI sample plate (Scout-MTP 384/600 AnchorChip; Bruker Daltonics, Bremen, Germany) prepared with a thin layer of the MALDI matrix CHCA. The TeMO worktable accommodates eight 96-well microtiter plates with peptide samples and two MALDI sample plates, each with 384 sample positions. The TeMO-96 pipetting head transfers 1 µL of each peptide sample from the microtiter plates to the MALDI sample plate where the droplets are allowed to dry. New racks of disposable pipette tips are delivered by the Tecan TeStack for each set of 96 tryptic extracts to avoid sample cross contamination. After the tryptic digest samples have dried, contaminants are washed away by dispensing 3 µL droplets of 0.1 % trifluoroacetic acid (TFA) onto the sample, followed by aspiration of the droplet after three seconds. Between washing of different sample spots, the pipette tips were rinsed using the Tecan washing station in a multi-step procedure included in the MALDI sample washing procedure. After emptying the tips, two cleaner steps with 90 µL MilliQ water each were employed, with soak

Dr. Niklas Gustavsson*, Dr. Dieter Weichart, Dr. Johan Gobom, Max Planck Institute for Molecular Genetics, Berlin *now based at the Mass Spectrometry group, Dept of Plant Biochemistry, Lund University, Sweden

times of 1 second and flow through set at 80. The first step included three cycles, with change of 5 µL each, and the second step included two cycles without change.

Controlling carry-over during MALDI sample plate preparation The consumption of pipette tips during washing of sample spots on the MALDI sample plates can be minimized by using the same set of tips for washing of all samples on an entire sample plate, as long as the pipette tips can be rinsed sufficiently between washing of different sample spots on the MALDI sample plate.

The efficiency of the pipette tip washing procedure was tested by preparing a set of three single peptides on adjacent sample spots on the MALDI sample plate (Figure 2), followed by washing the whole sample plate with one set of pipette tips.

Angiotensin I, neurotensin and ACTH 1839 were added onto quadrant Q1, Q2 and Q3 respectively. Nothing was added to quadrant Q4. The four quadrants were washed using the previously mentioned protocol with two cycles of 3 µL of 0.1 % TFA each. As shown in the MALDI-TOF mass spectra in Figure 2, no carry-over of the peptide prepared on the previous sample spot could be detected on the following spot. None of the peptides prepared on quadrants Q1 to Q3 were detected in the mass spectrum from quadrant Q4. The preparation of the three single peptides was performed in 32 parallel sets, with no carry-over detected in any of the mass spectra.

Assessing reproducibility of preparation for MALDI-TOF MS The reproducibility of the automatic sample application and MALDI sample

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

plate washing procedures was tested by preparing the same set of 368 tryptic digests of human proteins excised from a two-dimensional gel onto two MALDI sample plates. The plates were processed in parallel, and spectra were acquired under identical conditions on an Ultraflex LIFT mass spectrometer (Bruker Daltonics, Bremen, Germany). Using the MASCOT software, the peptide mass fingerprint data were submitted to a database search in the Swiss protein database (http://www.expasy.org/sprot/), considering scores larger than 62 indicative of reliable identification (p<0.005). With these settings, 98 proteins could be identified in total, of which 78 (79.6 %) were identified from both MALDI sample plates (see Figure 3a).

Three samples appeared to yield contradicting identifications in the two preparations. Upon closer inspection, however, the spectra were found to contain peptide signals from two different proteins. In each sample, both proteins were identified, but with different rank. Hence no conflicting results were observed between the two parallel MALDI sample plate preparations, indicating high reproducibility of both robotic handling and mass spectrometric identification. For demonstration, Figure 3b shows a selection of representative pairs of spectra originating from the two MALDI sample plates.

11

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

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

1SRef

3000

2500

1SRef

1500

1250

2000

1000

1500

750

1000

500

250

500

0

0 3000

1SRef

1SRef 1200

2500 1000

Summary In conclusion, the results presented here show that MALDI sample plate preparation using the Tecan TeMO unit allows robust, reproducible, simple and highly parallel analysis of protein digests.

They also demonstrate that the TeMO washing station saves pipette tips during the MALDI sample plate washing procedure, and that TeMO dispenses small volumes of protein digests with sufficient reliability to enable high throughput application of MALDI-MS based peptide mass fingerprinting.

2000 800

7

78

13

1500 600

1000

400

200

500

0

0 2400

Figure 3 (a): Overlap between protein identification results from two parallel MALDI runs with target plates supplied with identical samples using the Tecan TeMO module: Proteins identified on the first plate symbolized by red colour, and those identified from the second plate by blue colour. The number of proteins identified is shown below. The overlap from a total of 98 identified proteins was 79.6 %, without any conflicts of identification.

2600

2800

3000

1600

m/z

3200

1650

1700

1750

1800

1850

m/z

1900

1SRef

4000

1SRef 1500

1250 3000

1000

4000 750

500 1000

250

0

0

1SRef

1SRef

3000

1200

2500

1000

2000

800

1500

600

1000

400

500

200

0

0 1450

1500

1550

1600

1650

1700

1750

m/z

1600

1650

1700

1750

1800

1850

1900

m/z

(b): Examples of corresponding sections of mass spectra obtained for identical samples on the two MALDI targets (first target: red spectra, second target: blue spectra) Tecan Journal 1/2005

12

DETECTION

TM

GENios Pro

Dr. Gerlinde Zerza-Schnitzhofer, Dr. Manfred Lansing, Mag. Marieta Gueorguieva1, Mag. Carmen Ranftler and A.o.Univ. Prof. Dr. Jozefa GadekWesierski1

– the multi-functional reader proves a hit for multiplexing

Tecan Austria GmbH, Grödig, Austria and 1Medical University of Vienna, Vienna, Austria

Multiplexing assays for any applications offer a range of benefits over standard assays. In addition to being amenable to high throughput, they also allow the measurement and analysis of more than one parameter simultaneously and save time and money by conserving precious compounds and cell culture reagents.

However, for multiplexing to really work, there is the challenge of developing homogenous assays that do not require washing and sample handling steps, and the requirement of excellent performance, flexibility and multifunctionality from the detection instrument used.

Prof. Dr. Gadek-Wesierski and her research team at the Medical University of Vienna have shown that the GENios Pro multifunctional microplate reader from Tecan meets all these criteria and has a number of features such as individual optics for all reading modes that make it particularly suitable for multiplexing of cell-based assays with various readouts .

The team in Vienna aimed to multiplex two cell-based assays having fluorescent Tecan Journal 1/2005

and luminescent output to obtain information regarding viability and apoptosis directly from the same sample well, using Cisplatin (CP) as a test substance for treating HeLa cells. CP is a widespread anticancer drug used for chemotherapy. It inhibits viability and proliferation and induces apoptosis in human cervical carcinoma cells (see Figure 1).

In the experiment, HeLa cells S3 were cultured in 96-well plates and treated with increasing concentrations of CP, the CellTiter-Blue® Cell Viability Assay from Promega was used to calculate viability.

This assay determines the number of metabolically active cells based on the direct addition of resazurin to cells in a culture medium, which is then converted into resorufin by the cells. Measurements

Figure 1: Detection of apoptotic HeLa cells.

Untreated HeLa cells and HeLa cells treated with 40 µM cisplatin (15 hours) were fixed with methanol and stained with a fluoresceinconjugated monoclonal mouse antibody (M30-CytoDeath, Roche, Vienna, Austria). The CytoDeath antibody is specifically for caspase-3 cleaved cytokeratin-18 and stains cells in various stages of apoptosis. Cell nuclei were made visible using DAPI. Mitotic (M) and apoptotic (A) cells are marked with the following symbol ->.

C U STO M E D R ESTUEPCPTO I ORT N

were taken after the appropriate incubation period using the GENios Pro reader (Figure 2). Resorufin is activated at 545 nm and emission is detected at 590 nm. The fluorescence reading can then be correlated with the number of metabolically active, living cells, if appropriate serial dilutions of cells have been performed accordingly.

In experiments with adherent cells, excitation and detection through the bottom of a microplate are preferable to top reading as this reduces disruptive influences of the culture medium on the measurement signal. A preliminary test with CellTiter-Blue® showed that the detection limits in bottom reading mode were between six and eight times lower than in top reading mode. This means that fewer cells and materials can be used to achieve the same results, which is particularly advantageous for cell-based applications.

Prof. Dr. Gadek-Wesierski and her research team are based at the Institute of Cancer Research at the Medical University of Vienna. The institute is focused on basic cancer research, scientific education and the development of a new understanding of cancer disease.

The group is studying the mechanisms of cell cycle regulation during malignant transformation of cells. The expression, activation and inhibition of tumor suppression proteins (eg, p53),

Figure 3: Multiplex analysis of viability and apoptosis in HeLa cells. Viability was analyzed using CellTiter-Blue® Assay and apoptosis using Caspase-Glo 3/7™ Assay following incubation of the cells in 96-well microplates with 0 – 40 µM cisplatin (CP).

Figure 2: GENios Pro microplate reader. Features such as rapid fluorescence bottom reading and reagent addition by injectors make this multimode reader particularly suitable for a large number of cell-based assays in HTS.

Figure 3 shows the results of the CellTiterBlue® assay after the HeLa cells were incubated for 24 hours with various concentrations of CP (0, 1, 5, 20, 40 µM).

As the CP concentration increases, the number of metabolically active cells is reduced by approximately 70 %.

Information on the apoptosis rate was obtained in the multiplex assay using the Promega Caspase-Glo 3/7™ assay to measure the activity of caspase-3 and -7, both of which play a key role in the apoptosis of mammalian cells. Caspase activity is measured by adding a preluminescent substrate solution into

the sample wells containing the CellTiterBlue reagents, after having taken the viability readings. The substrate solution contains a stabilized luciferase and is optimized for caspase/luciferase activity and cell lysis. The substrate solution destroys the cells, the preluminescent substrate is cleaved by active caspases releasing luciferin that is then consumed by the luciferase. The resulting luminescent signal is proportional to caspase activity and can be measured.

The results in Figure 3 show a clear increase in apoptosis at high CP concentrations. The number of apoptotic cells increased in this experiment by approximately four fold. Correlating the readings from both the viability and apoptosis analyses with the number of cells allows the percentage of apoptotic cells to be determined.

Summary

which play a key role in cycle control, are investigated in different cell lines and species. Additionally, the group is studying the effect of new anti-cancer drugs, in particular regarding their potential for reactivating the tumor suppressor protein p53, which results in enhanced efficiency of chemotherapy treatment. http://www.p53parp.at complex cellular processes. Additionally, multiplexing offers the advantage of reducing experimental variation, like fluctuations in cell numbers due to cell clumping and dispensing. Homogenous assays, which require no washing and separation stages, can easily be integrated into Tecan Robotic systems with the GENios Pro Reader for use in high throughput screening. Stages such as the addition of various assay reagents by injectors into 96 and 384 well plates, incubation at 37° C and plate-shaking, can all be carried out in this modular reader. The measurement methods available on the GENios Pro are suitable for a large number of multiplex assays and range from absorption and fluorescence (FI, FRET, TRF) to various luminescence techniques (BRET2™, Glow and Flash Type Luminescence).

This experiment combined two cell-based assays in one multiplex experiment but, using additional assays, e.g. LDH release or reporter gene assays, the team believes that multiplexing could easily be extended to answer further questions.

Acknowledgement We would like to thank Prof. Dr. GadekWesierski and her team at the University of Vienna for providing the data and Dr. Katarina Bohm and Dr. Heinz Winkler from Promega for kindly providing the reagents.

GENios Pro™ is a trademark of Tecan Group Ltd. Cell Titer-Blue® and Caspase-Glo 3/7™ Assays are trademarks of Promega corporation.

BRET2™ is a proprietary technology from BioSignal Packard.

Patents pending.

Multiplexing is an efficient way of obtaining more than one set of experimental data from the same sample well in order to better understand Tecan Journal 1/2005

13

14

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

Just a friendly phone call away

Tecan Journal 2005

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

Laurent Nussbaumer is Supporting Engineer at Syngenta’s Crop Protection Research facilities based in Basel and Stein in Switzerland.

Mr Nussbaumer is responsible for equipment at both sites and has worked with Tecan on several projects over a period of seven years.

What is the main role of your research facilities?

As a whole, the Crop Protection Research Facilities are responsible for investigating chemicals that have the potential to protect crops, mainly fungicides, insecticides and herbicides. The site in Basel is essentially a dispensary, where all the research compounds are stored, prepared into solution and pipetted by Tecan systems into reaction microplates.

These plates are then transferred to the facility in Stein where we perform a range of preliminary and validatory tests, also using Tecan systems, on the chemicals themselves, and on the effects they have on plants in different formulations and concentrations.

What level of sample throughput are you achieving?

At the moment our throughput is around 200-300 compounds per week, not what I would call high throughput at all, but we are performing numerous tests at many different concentrations with each individual compound.

What Tecan equipment do you have and how long have you been using it?

We have been using Tecan equipment since we started operations seven years ago. At the Basel site, we have two GENESIS RSPs with liquid handling (LiHa)

arms on each for pipetting. A Freedom EVO and carousel handle the plates so the process is completely automated. In Stein we have GENESIS RSPs equipped with LiHa and robotic manipulator (RoMa) arms, carousels and specially adapted needles that spray the chemicals onto the plants.

Did Tecan provide you with application support to set up these processes?

Yes, we have had application support from the beginning. At first, our equipment was set up for high throughput screening but every time we have a new project or change the process we have needed to adapt the equipment.

Sometimes we can make the changes ourselves, but if we need instrument upgrades or other alterations we always directly involve Tecan.

And how do you rate Tecan’s customer service?

Very good! First and foremost, the equipment is generally very reliable.

However, whenever there is a problem, the Tecan engineers guide us through some general checks so that they can fully analyse the problem and advise whether there is some way we can resolve the problem ourselves. If not, they are with us within 24 hours or if possible, because we are quite close, within 3 hours. Tecan’s good customer service is very important to us.

Tecan Customer Support Whether the customer needs assistance in reliable automation, precise and safe liquid handling, or maintenance and operation of robotics and detection products, services are in place to assist the customer at all levels of activity. In this way, Tecan is able to offer the expertise necessary to ensure professional use and high-performance operation of Tecan equipment.

Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.com Photography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net Editor: kdm/UK www.kdm-communications.com Print: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Switzerland AG, Marketing Communication, Seestrasse 103, CH-8708 Männedorf, journal@tecan.com, www.tecan.com ©2005, Tecan Trading AG, Switzerland, all rights reserved Tecan Journal 1/2005

15

16

GLOBAL N EWS

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

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

AACC

Orlando, FL

July 24-28 2005

Drug Discovery

Boston, MA

August 7-12 2005

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

Japan

USA

December 10-14 2005

Australia 2. ComBIO 2005

Adelaide

September 25-29 2005

Asian Pacific Congress of Clinical Biochemistry

Brisbane

September 14-18 2005

Austria +43 62 46 89 33 Italy +39 02 215 21 28

Belgium +32 15 42 13 19 Japan +81 42 334 88 55

Sweden +46 31 75 44 000

Tecan Journal 1/2005

China +86 10 586 95 936

Denmark +45 70 23 44 50

Netherlands +31 18 34 48 17 4

Switzerland +41 44 922 89 22

UK +44 118 9300 300

Portugal +351 21 000 82 16 USA +1 919 361 5200

France +33 4 72 76 04 80 Germany +49 79 51 94 170 Singapore +65 644 41 886

Spain +34 93 490 01 74

Other +43 62 46 89 33

www.tecan.com