SpectraMax® L multi-detector microplate luminometer - Molecular ...

Increase your luminescence
throughput:
SpectraMax ® L
multi-detector microplate
luminometer
2009 Drug Discovery User Forum
Cathy Olsen
Application Scientist
MDS Analytical Technologies (US) Inc.
April 25, 2009

Agenda
• Introducing the SpectraMax ® L microplate luminometer
o Multi-detector options
o Features
• Luminescent G-protein coupled receptor assays with aequorin
o Features & advantages of aequorin GPCR assays
o Performance on SpectraMax L microplate luminometer
o Scale up to FLIPR TETRA® system with aequorin option
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Introducing the SpectraMax ® L luminometer
• PMTs are optimized for typical wavelengths of luminescent emission
• Ultra-fast photon counting technology
o Amplifies PMT signal with fast pulse amplifier
o Reduced noise
• Higher signal-to-noise ratio
• Low crosstalk
• MaxRange for 9 orders of
magnitude
o Simultaneous analog and
photon counting capability
o No need to dilute bright samples
o Excellent sensitivity for dim
samples
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How luminescence is detected
• Photomultiplier tube (PMT) converts incoming photons into electrons
• Incoming photon strikes photocathode → generates electron
• Electron flows through a series of electron multipliers (dynodes) to the anode
• Current flowing from anode ∝ number of photons at the photocathode
• Amount of amplification a PMT can produce depends on (a) number of
dynodes and (b) voltage applied to it
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Source: http://micro.magnet.fsu.edu/primer/digitalimaging/concepts/photomultipliers.html

SpectraMax ® L luminometer: Four PMT settings
• Analog Only: Ideal for very bright signals above 1.6 x 10 7 RLU (low PMT voltage)
• Photon Counting: Ideal for very dim and medium signals below 25 x 10 6 RLU (digital)
• AutoRange: Extends range of Photon Counting mode by adding Analog (high PMT
voltage)
• MaxRange: Combines AutoRange and Analog Only modes
o Captures entire dynamic range
o 10 to 10 9 RLU
AutoRange
MaxRange
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PMT calibration
• Automatic PMT calibration
o Uses internal light-emitting diodes (LEDs)
• Manual PMT calibration
o Uses 4 wells containing actual assay material
• Manual Set option
o Manually enter calibration factors for each PMT
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Results of PMT calibration using internal LEDs

SpectraMax ® L luminometer - configurable
• With injectors → flash luminescence
• Without injectors → glow luminescence
• Configurations to meet every user’s
needs
o Single channel (PMT)
• 0 or 2 injectors
o 2-channel
• 0, 2, or 4 injectors
o 6-channel (PMT)
• 0 or 12 injectors
• Upgradeable
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Higher-throughput luminescence assays
• Example: typical dual-luciferase assay
• 2 injections + 2 reads = 25 seconds per well
Number of PMTs/injector pairs 1 2 6
Time to read a 96-well plate 40 min 20 min 6.7 min
Plates read in 8 hours 12 24 72
StakMax ® plate handling system
enables automated reading of up
to 50 plates.
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SpectraMax ® L luminometer + StakMax plate handling system

AutoWash function
Upon instrument shutdown:
• Injectors reverse, returning reagents to bottles
o Saves reagents, money
• Injectors are automatically washed
• Prevents precipitation from clogging injector lines
o Fewer maintenance worries
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SoftMax ® Pro software
• Data acquisition, analysis, graphing
• Supports MDS AT microplate readers
• Includes >120 pre-configured protocols
o Optimal reader settings for each assay
o Assay-specific data analysis
Need to show GLP/GMP compliance with FDA 21 CFR Part 11 requirements
• SoftMax Pro GxP provides FDA 21 CFR Part 11 compliance tools
o Electronic signature support for verification, authorization, and approval
o Audit Trail to track and record user and administrator actions
o And much more…
• Optional software validation tools
• Visit http://www.moleculardevices.com/pages/software/softmax.html
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Applications for SpectraMax ® L system
• Aequorin GPCR assays
• Luciferase reporter assays, including DLR (Promega)
o Flash & glow versions
• Bioluminescence resonance energy transfer (BRET)
o BRET1 & BRET2 options
• Cell viability and toxicity assays
• Mycoplasma monitoring
• ATP detection
• Luminescent ELISAs
• Acridinium ester
• And many, many more…
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Luminescence protocols in SoftMax ® Pro software
• Aequorin GPCR
• Dual-Luciferase Reporter (Promega)
• Dual-Glo Luciferase (Promega)
• Ready-To-Glow Secreted Luciferase (Clontech)
• Luminescent ELISA
• CellTiter-Glo (Promega)
• MycoAlert (Lonza)
• ATP assay
• dsDNA assay
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Moving on…
• Introducing the SpectraMax ® L microplate luminometer
o Multi-detector options
o Features
• Luminescent G-protein coupled receptor assays with aequorin
o Features & advantages of aequorin GPCR assays
o Performance on SpectraMax L microplate luminometer
o Scale up to FLIPR TETRA® system with aequorin option
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Aequorin overview
• Photoprotein originating from the jellyfish Aequorea victoria
• Aequorin binds Ca ++ → oxidizes coelenterazine → CO 2 + light
• Aequorin is used to detect GPCR signaling via Ca ++ in cells
• Reaction is very fast (10-30 seconds)
• Assay is read on luminometer equipped with injector or pipettor
GPCR
Agonist
α β γ
PLC
Apoaequorin
Coelenterazine
IPs
Aequorin
+
Ca ++
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Flash lumi

Advantages of aequorin assays
• Luminescent signal reduces problems of auto-fluorescence,
compound interference
• Targeting photoprotein expression to specific parts of cell allows
more target-specific signal resolution
• Wide dynamic range (10 -7 to 10 -4 M) covers physiological Ca ++
signals
• Low calcium buffering effect compared to fluorescent reagents
• Expression is non-toxic
• Reduced cost (10x less) compared to fluorescence or luciferase
assays
• Cells can be delivered in suspension*
o Reduces cost
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o Increases throughput *These assay methods are also described in the literature, e.g. in Boie et al., Eur. J.
Pharmacol., 340(2-3):227-241 (1997), and in United States Patent 6,872,538 and
European Patent 1,145,002. Users interested in the patented methods may wish to
consult legal counsel in evaluating these patents.

Disadvantages
• Requires reconstitution with coelenterazine
o Similar to dye loading, but from 4 hours to overnight
• Aequorin consumption
o No long-term recording or multiple additions
• Possible EC 50 /IC 50 shift compared to other methods
• Not all GPCR assays are amenable to aequorin
o GPCRs that do not signal via Ca ++
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Aequorin assays on SpectraMax ® L system
• Preconfigured protocol in SoftMax ® Pro software
• Automatic data reduction and analysis
• 4-parameter curve fit
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AequoZen frozen cells (PerkinElmer)
• Cryopreserved, γ-irradiated cell lines
• Good for single use, cannot be propagated
• CHO-K1 cell lines expressing GPCR
o Histamine H 1 (stronger response)
o Muscarinic M 5 (weaker response)
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AequoZen M 5 cell line: agonists
Concentration-response curves from SpectraMax ® L system
EC 50 from product insert = 16.2 nM
EC 50 from product insert = 25.7 nM
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AequoZen M 5 cells - antagonist
Concentration-response curves from SpectraMax ® L system
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IC 50 from product insert = 3.2 nM

AequoZen H 1 cell line - agonists
Concentration-response curves from SpectraMax ® L system
EC 50 from product insert = 4.6 nM
EC 50 from product insert = 4.8 uM
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AequoZen H 1 cell line - antagonists
Concentration-response curves from SpectraMax ® L system
EC 50 from product insert = 4.9 nM
EC 50 from product insert = 2.8 uM
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SpectraMax ® L & FLIPR TETRA® systems
• SpectraMax L provides low to medium throughput for luminescent assays
o Assay development
o Initial screens
o StakMax ® plate handler for increased automation
o Aequorin results similar to FLIPR TETRA
• FLIPR TETRA provides high-throughput capability
o Aequorin assays in suspension or adherent modes
o 1536-well assays
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Conclusions
• SpectraMax ® L system is highly configurable & upgradeable
• Increases throughput for luminescence assays
o Especially when paired with StakMax ® plate handling system
• Enables a vast array of luminescent applications
• SoftMax ® Pro software with preset protocols
• Data quality comparable to FLIPR TETRA®
o SpectraMax L system for low-medium throughput
o FLIPR TETRA for high throughput
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Acknowledgments
• Carole Crittenden
• Michael Su
• Wilson Toy
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Thanks for your attention
• For more information online:
http://www.moleculardevices.com/home.html
• Poster #P8007: Session P8 Automation and Detection Platforms
• SpectraMax L demos will be presented in the MDS AT booth
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