TriStarLB 941Multimode MicroplateReader

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TriStar LB 941

Multimode Microplate Reader

Dedicated Optical Path System (DOPS)The TriStar operates with a dedicated optical path system (DOPS) like the Mithras: for each readingtechnology there is an optimum optical system guaranteeing high sensitivity and wide dynamic rangefor all measurements. The path selection is done manually by the user according to the reading tech-nology needed. The switching between the opticalpaths is extremely easy without the need for any toolsand re-alignment andcan be executed withinseconds. Detection limits of lessthan 6 attomol ATPand 3 zmol Firefly areimpressive examplesfor the outstandingperformance.

Sensitivity and EconomyThe TriStar LB 941 multimode plate reader for absor-bance, luminescence and fluorescence is unique in itsreliability and sensitivity. In addition to the 3 majorreading technologies representing more than 75 % ofmicroplate assay performed TriStar offers BRET andBRET² detection technology. With the choice of up to 4 reagent injectors for fast and ultra-fast flash reactions as well as temperature control for long-term cellular assays the TriStar meets the demandsof today´s and tomorrow´s research.Inspite of its outstanding performance and combin-ation of features the TriStar represents an economic-ally attractive solution.

absorbance

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luminescence

TriStar Microplate Readers

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Selected Low Noise DetectorsIn addition to optimized optical paths selected highquality detectors are key to excellent sensitivity. The photomultiplier tubes (PMTs) used in the TriStarmultimode readers are operated in the state-of-the-art ultra-fast single photon mode. Stringent selectionalong narrow limits for lowest background and highest efficiency ensures reliable and superior performance throughout all instruments.For very demanding applications the photomultipliertubes can be equipped with a Peltier-type cooling unitreducing the noise even further.

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These filter carriersare software-drivenand provide quickchange of emissionand excitation filterswithin one run espe-cially important forall applications with ratiometric readout, e.g. Indo-1, Fura 2 or BRET.The changing time for adjacent filter positions is aslow as 130 ms made for getting highest resolutionin kinetics.

The filters offered by BertholdTechnologies are high quality filters with selected transmissionand bandwidth specifications forthe respective applications. Additional filters can by addedby the users at any time.

Filters – the better choice for most applicationsThe TriStar LB 941 is a filter-based system to extractthe greatest benefits from the Dedicated OpticalPath System (DOPS) as only filters with high transmission characteristics ensure best sensitivity. Especially BRET/BRET² and other colour lumines-cence applications are only possible with an extremely efficient optical system together with appropriate filters as used in the TriStar readers.Mithras microplate readers can work with up to 20 excitation and more than 30 emission filters conveniently mounted in exchangeable slides andwheels.

Calcium MonitoringIntracellular Ca++ levels have become important indicators for the activation state of ion channelsand G-protein coupled receptors as well as for thephases of apoptosis and cell injury. Though the respective kinetics and the absolute amounts of theCalcium levels are different for each of these physiological processes there are common ways formonitoring them.

Luminescent labelslike Aequorin as wellas fluorescent onesare versatile andwidely used solu-tions for microplateassays. Fura 2 andIndo-1 provide a ratiometric readoutthereby reducing

effects caused by leaking or bleached dyes or varyingassay conditions.

OregonGreen offers a fastsingle wavelength readoutespecially suited for highthroughput environments.These assays can easilyand reliably be performedwith the use of the TriStar´svariable reagent injectors.

ATP DeterminationA detection limit ofless than 6 attomolof ATP per wellmakes the TriStarone of the bestsuited microplateinstruments for the determination of

cell viability, e.g. in tumor chemosensivity assays, cell proliferation, antibiotic susceptibility testing or hygienemonitoring.

DNA Probe AssaysSeveral diagnostic DNA probe assaysbased on Acridinium ester labelled oligo-nucleotides are commonly used providingthe most sensitive detection and diagnosisof infectious diseases.

Fluorescence Resonance EnergyTransfer (FRET)

Fluorescence Res-onance EnergyTransfer (FRET)has for long beenknown to be asmart method forvisualisation ofmolecular interac-tions of proteinsand nucleic acids.

The discovery and further development of the Fluorescent Protein family as well as the Cy dyes haveled to an increased use in microplate assays. Anotherpopular donor/acceptor combination is FITC and Rhodamine.

FRET is based on the fact that a donor dye like CFP inan excited state can transfer a part of this energy andexcite an acceptor dye like YFP. Its emission can be detected as soon as both dyes are in close proximity.

Reporter Gene AssaysIn basic research of gene regulation aswell as in drug discovery the use of luciferases, ß-galactosidases, ß-glucuronidases and secreted alkalinephosphatases have become a standard

tool offering thehighest sensitivity.Especially the dualluminescence typeassays, e.g. Dual-Luciferase® Re-porter Gene Assay,have become afavourite means asthey provide an internal control for transfection efficiency or generalexpression level. Superior sensitivity in lumines-cence – better than most dedicated luminescenceplate readers – make the TriStar the instrument ofchoice for reporter gene assays with a detectionlimit of less than 3 zeptomol Firefly luciferase.

TriStar LB 941

Multimode Microplate Reader

Homogeneous Functional GPCRAssay Using BRET DetectionG-protein coupled receptors, also referred to as 7-transmembrane (7TM) receptors, comprise thelargest and most diverse superfamily of proteinsknown. To a minor part only the ligands are known.

Especially in the field of G-protein coupled receptorresearch, the BRET technology offers the opportunity for the establishment of a homogeneousand universal functional assay, taking advantage of the fact that ß-arrestin (which is naturally playinga role in the desensitisation of the receptors) bindsto the intracellular part of the activated receptor.

Renilla luciferase emits blue lightat 480 nm upon addition of itssubstrate coelenterazine.

Activation of receptor (conform-ation change, attachment of G-protein) due to binding of ligand.

ß-arrestin / eYFP fusion protein at-taches to receptor enabling energytransfer between Renilla luciferaseand the eYFP moiety resulting in arising peak of green light at about 530 nm.

BRET is based on the fact that the energy derived froma Renilla luciferase reaction can be used to excite a fluorescent protein molecule if the latter is in close proximity to the luciferase enzyme. There are severaladvantages of BRET over other methods.

It is a non-radioactive and homogeneous tech-nology. The ratiometric signal minimises inter-ferences from assay conditions and keep the time management non critical. There is no auto-fluorescencecoming from compounds or cell and buffer components

as no light sourceis required.

Effect of agonist AVPon COS7#41 cellsexpressing VP2-Rlucand ß-arrestin-GFP2

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Luminescent Immunoassays (LIA, ILMA)

By exchanging colori-metric substrates ofhorseradish peroxidaseor phosphatases withluminescent ones a100-fold increase insensitivity can usually be achieved. MikroWin

software with the curve fitting option adds convenientand extensive data evaluation capabilities to the superb instrument performance.

Caspase AssaysMonitoring the activity of Caspases – a group of cystein-aspartic acid peptidases – is a key method inapoptosis research. The assays are designed aroundspecific peptide substrates for Caspase 3, 7, 8 and9 respectively which will be cleaved when Caspasesare present indicating cells are in an apoptotic state.

Assay technologiesare available withluminescent read-out through the release of Luciferinwhich acts as thesubstrate for the

subsequent lightgenerating Lu-ciferase reaction.Other setups re-lease fluorescentdyes, e.g. Fluores-

cein or Coumarinderivatives, uponcleavage or colou-red substancesdetectable withabsorbance meas-urements.

TriStar LB 941

Multimode Microplate Reader

Temperature ControlThe TriStar can be equipped with a heating system forthe microplates for assays which need to be measuredat elevated and constanttemperatures, e.g. commonin cells based assays. Eachparameter file can have itsindividual temperature setting which is simply activated by loading the respective parameter file. The detector is cooled by a Peltier unit to keep its temperature and noise low and maintain its high sensitivity.

KineticsWith the Kinetic and Repeated measurement modestime courses of physiological processes can be moni-tored over short (few seconds) and long periods oftime, e.g. up to 7 days. Enzyme activities, phagocyto-sis and Calcium monitoring are typical applications forthe use of the kinetic functionality. The Kinetic and Repeated modes includepossibilities to define interval and cycle timesas well as intermittentinjections or dual labelsettings (as in BRET andFura 2 assays).

Flexible graphical displays and numerous calculation options enable sophisticated data evaluation, e.g. AUC,Vmax, slope, minimum, maximum. In addition the kinetic mode can support the user to establish optimumassay conditions or to monitor the reaction time ofluminescence or fluorescence assays and define appro-priate measurement times.

Microplate ShakingThe integral shaking function with 3 shaking modes –linear, orbital and double orbital – each of them adjustable in amplitude and speed can be activ-ated to enhance mixing and optimise sample distribution before measurement. This canbe very useful in long-termkinetic assays with cells.

Reagent InjectorsUp to 4 independently controlled injectors with variable volume give entire freedom in the selectionof assay type and assay sequence. The injectors are located in measurement and pre-measurementpositions of the luminescence and the fluorescencepath respectively.

JET injection tech-nology stands forprecision and accur-acy - better than 98 %-and the most reliable way ofproper mixing of theadded reagents.Tuneable injectionspeed covers the

different demands of viscosities, cells or filling heights.

The volumes are adjustablefrom 10 to 100 µL–in incre-ments of 1 µL. This is perfectly matching the requirements for reagentaddition into 96 and 384well plates. Dispensing caneven be performed within a running kinetic measure-ment, e.g. to watch the effects of added agonists /antagonists. Cell-friendlymaterials and diameters as well as the ability for lowspeed injection make the Berthold Technologies JETinjectors the ideal tools for the addition of cell suspensions to compound plates, e.g. in Aequorin-type Calcium assays. “Economy Prime” a special priming mode which isunique to Berthold Technologies JET injectors hasbeen developed and implemented to facilitate homogeneous filling of the injection lines whilstusing as little prime volume as possible helping theusers to save valuable reagents.

Small footprintTriStar LB 941 has been designed with a very smallfootprint. Housing dimensions are only 472 mm x494 mm including reagent injectors saving valuablelaboratory space.

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Well ScanningThe Scanning mode enables the researcher to scan awell of a microplate in up to 10,000 steps each of themcreating an individual data point. This procedure is recommended when the assay is based on adherentcells which are heterogeneously distributed and signalsare not located at the centers of thewells. A graphical representation ofthe signal distribution as well asmathematical formula for minimum,maximum, average and total signalfacilitate data interpretion e.g. inchemotaxis applications.

Microplate FormatsThe basic TriStar models accept any kind of microplateregardless of its colour and design. The accepted plateheight is 15 mm, the standard measure for all com-mon 96 and 384 well microplates. Microplates withheights of up to 20 mm, like most 24 well microplates,can be used in the model with variable plate height.In addition Petri dishes, Terasaki plates and filtermembranes can be loaded with respective adapters.

Plate StackerUnattended operation and increased throughput arefacilitated with the Stacker unit LB 931. The stackerunit can simply be attached to the TriStar without anymodifications of the main instrument.Two different types of stacks are available to hold either 25 or 50 microplates. They are easily exchan-geable to match everday´s varying throughput needs. The stacker accepts 24, 96 and 384 well microplates

from all major plate manufactur-ers. Manual and robotic loading isstill possible even with the stak-ker attached by simply removingthe plate stacks. A unique featureof the LB 931 Stacker is the re-stacking operation mode whichcan be used in long-term kineticmonitoring of samples in mult-iple microplates, e.g. circadianrhythms of gene expression.Barcode readers may be added

and used for positive plate identification or for auto-matically selecting parameter files containing the res-pective measurement settings. The software supportsmultiple options for data storage using the barcode information.

MikroWin 2000 SoftwareThe Windows® based software combines operationand definition of instrument settings as well as datareduction and evaluation.

The wells for measurement and injection can be selected independently. Various operations can bepicked and placed in any order to accommodate for all different requirements of the assays to be performed.

Kinetic data reduction andgraphical display of the res-pective curves help the userto judge the results.

Any type of ratio calculation,e.g. DLR®, or other mathe-

matical formulas can be linked to every well individ-ually.

Standard curve fitting and calculation of unknown samples is available for those users looking for

quantification of their re-sults (Advanced I and II).All data can be exportedin EXCEL® or ASCII formats.

21 CFR part 11 Statement System access is controlled by log-on routines whereunique combinations of user name and password arerequired to enter operation levels.

Unauthorised access is denied and documented.

Electronic signatures are provided by user ID anddate/time stamps in data files.

Files can be viewed in electronic and printable form.Audit trails are generated.

System has been developed, tested and documented according to ISO 9001 regulations.

MikroWin 2000 Advanced versions are 21 CFR part 11 compliant

TriStar LB 941Technical Specificationand Order Information

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Detection Units Low-noise photomultiplier tubein single photon counting mode,spectral range: 340 - 650 nm; photo diode: 200 - 1000 nm

Excitation Sources halogen lamp, 75 W (340-700 nm);Measurement LuminescenceTechnologies Top-reading Fluorescence

AbsorbanceBRET (option)

SensitivityLuminescence <6 amol ATP; <3 zmol firefly luciferase,

equals less then 800 moleculesFluorescence <2 fmol FITCAbsorbance linear range 0-3.5 OD

accuracy <2 % cv (@ 2.5 OD)Dynamic Range >6 orders of magnitudeCrosstalk low crosstalk through

crosstalk reduction design: 5 x 10-6

Injection Unit up to 4 injectors (variable volume:10–100 μL), JET Injection technologyinjection into 384 well plates

Plate formats 6 to 384 well microplates with outerdimensions 86.0 x 128.2 mm (WxL)height of 15 mm (+/-1 mm) in stan-dard models; height of 14 – 20 mmin model with variable plate height

Temperature Control +5°C above RT to 42 °C (option),includes cooling unit for PMT

Robot BUTLER LB 930 (option)Stacker Stacker LB 931 (option)Interface serial RS232, 9 pinPC Operating System Win 98, Win 2000, Win NT,

Win XP, Win VistaPC Requirements Pentium processor, 500 MHz

(or better), CD ROM drive, display1024x768 (or better), serial port,parallel port, USB

Software MikroWin 2000Power Supply 110 - 240 V; 50 /60 Hz; 240 VARegulations CE, UL, CSATemperature Range storage: 0° - 40° C

operation: 15° - 35° CHumidity 10 – 85%, non condensingDimensions 472 x 494 x 374 mm

(WxDxH), incl. injectorsWeight 40 kg

Operation ModesIntegral measurement 0.1 – 600 secKinetics measurement total time up to 24 h Repeated measurement total time up to 7 daysPlate repeats up to 50,000Delay up to 600 secScanning up to 10,000 individual meas. pointsDispensing with 4 independently controlled

variable volume injectorsShaking 3 modes, variable amplitude and speed

Order Information Order number

TriStar: Luminescence, Fluorescence (top), Absorbance, MikroWin Lite* 49178-10

TriStar T: Luminescence, Fluorescence (top), Absorbance, temperature control, cooled PMT, MikroWin Lite* 49178-15

TriStar i: Luminescence, Fluorescence (top), Absorbance,injector 3+4, MikroWin Lite* 49178-20

TriStar Ti: Luminescence, Fluorescence (top), Absorbance,temperature control, cooled PMT, injector 3+4, MikroWin Lite* 49178-21

TriStar vTi:Luminescence, Fluorescence (top), Absorbance,temperature control, cooled PMT, injector 3+4,variable plate height, MikroWin Lite* 49178-25

Injector 1 pre-position, 10-100 µL 37772-11Injector 2 measurement position L, 10-100 µL 37772-12Injector 3 measurement position L, 10-100 µL 37772-13Injector 4 measurement position F, 10-100 µL 37772-14BRET package: filters for BRET and BRET2 39350Chroma-Glo package: filters for Chroma-Glo™ 43544Additional excitation filter slide “B̋ 41537Additional excitation filter slide “C̋ 41538Additional emission filter wheel 38882Luminescence test plate for QC 40105-10Luminescence test kit for QC 45366Absorbance test plate 43513Cleanit Daily, injector cleaning solution 45218Microplates 96 well, white, 100 pcs 23300Microplates 96 well, black, 100 pcs 23302Microplates 96 well, white, sterile, 50 pcs 51838Microplates 96 well, black, sterile, 50 pcs 51839Micropl. 96 well, white, clear bottom, cell culture, 100 pcs 24910Micropl. 96 well, black, clear bottom, cell culture, 100 pcs 38840Microplates 384 well, white, 40 pcs 32505

*Upgrade (MikroWin Advanced versions I and II) and additionallicences available.

For further information of available filters see Filter Data SheetDefault parameter files, e.g. BRET, BRET2, Dual-Luciferase® and Dual-Glo™ reportergene, Chroma-GloTM, Fluorescein, Umbelliferone, FRET, kinetics and repeated type,ratiometric assays.

Patents: EP 0 523 522 ; EP 1 279 946 (pending); DE 4 123 818 ; DE 10 136 863(pending); 6,949,754 US

DLR®, Dual-Luciferase®, Dual-Glo® and Chroma-GloTM are registered trademarks of Promega.

BERTHOLD TECHNOLOGIES GmbH & Co. KG

P.O. Box 100 16375312 Bad WildbadGermany

Phone: +49 7081 177-0Fax: +49 7081 177-100E-mail: Bio@Berthold.comInternet: www.Berthold.com/bio