cyte applications

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Drug Discovery Labcyte Deerac™ and Echo ® liquid handling systems deliver better scientific data, cost savings and reduced waste for applications throughout the drug discovery process, including compound management, biochemical and cell-based screening, siRNA screening and assay development. Cost Savings in Compound Management and Screening With 2.5 nL transfer volumes, Echo liquid handling systems enable miniaturization of compound storage libraries and screening assays into 384-, 1536- and even 3456-well plates. ADE transfer is tipless and touchless, saving Echo users significant costs in tips, plates, wash solutions and waste disposal. Case Study - Echo Liquid Handlers Enable 3456-Well Assays Merck Frosst Centre for Therapeutic Research (Quebec, Canada) uses the Echo 550 liquid handler to transfer their 384-well compound library storage plates into 3456-well assay plates. This miniaturization led to a tremendous savings in reagent and compound usage, as well as in reduced plate handling requirements. More Accurate Primary Screening Echo liquid handling systems simplify and optimize plate preparation for a wide range of compound and siRNA screening assays. Our unique Echo Dose- Response application software eliminates serial dilutions, creating precise multi-log dose-response curves in just three steps. Tipless transfer ensures that sticky compounds are not lost on tips and plates, yielding more accurate results so that you don’t miss your best hits!

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Page 1: Cyte Applications

Drug DiscoveryLabcyte Deerac™ and Echo® liquid handling systems deliver better scientific data, cost savings and reduced waste for applications throughout the drug discovery process, including compound management, biochemical and cell-based screening, siRNA screening and assay development.

 

Cost Savings in Compound Management and Screening With 2.5 nL transfer volumes, Echo liquid handling systems enable miniaturization of compound storage libraries and screening assays into 384-, 1536- and even 3456-well plates. ADE transfer is tipless and touchless, saving Echo users significant costs in tips, plates, wash solutions and waste disposal.

Case Study - Echo Liquid Handlers Enable 3456-Well AssaysMerck Frosst Centre for Therapeutic Research (Quebec, Canada) uses the Echo 550 liquid handler to transfer their 384-well compound library storage plates into 3456-well assay plates. This miniaturization led to a tremendous savings in reagent and compound usage, as well as in reduced plate handling requirements.

More Accurate Primary ScreeningEcho liquid handling systems simplify and optimize plate preparation for a wide range of compound and siRNA screening assays. Our unique Echo Dose-Response application software eliminates serial dilutions, creating precise multi-log dose-response curves in just three steps. Tipless transfer ensures that sticky compounds are not lost on tips and plates, yielding more accurate results so that you don’t miss your best hits!

Page 2: Cyte Applications

  Figure 2. Two-fold 16-point titration curves of compounds H89 and H9 assembled from the LOPAC collection (Sigma-Aldrich) using an Echo 555 liquid handler. Kinase assay IC50 values were comparable to previously published data.  

Case Study – Echo Liquid Handlers Deliver Fewer False Negatives Bristol-Myers Squibb demonstrated fewer false negatives in IC50 assays prepared using the Echo 550 liquid handler versus traditional aqueous serial dilutions. The results revealed 110 compounds that showed significant activity when diluted using ADE, but would have been eliminated as ineffective when diluted using traditional serial dilution. Closer examination revealed that the false negatives are often due to the loss of hydrophobic compounds adhering to pipette tips and dilution vessels. Without their Echo liquid handler, BMS might have missed their best leads.  

Figure 3.  Compounds in the lower left quadrant have low IC50 values using both dilution methods and are considered active. Compounds in the upper right quadrant have high IC50

values using both methods and are considered inactive. 10% of the compounds tested (lower right quadrant) were mistakenly identified as biologically inactive using traditional serial dilution, but ADE transfer revealed potentially strong leads. The circled compounds had IC50

values that were 10- to 100-fold lower when transferred using ADE than when transferred with pipette tips and serial dilution.  

 

 

Case Study – Deerac HTS Enables Miniaturized Enzyme Inhibition Assays The Deerac™ HTS system enables miniaturization for a wide range of biochemical assays. Using the Kinase-Glo® (Promega) enzyme inhibition assays, we reduced the assay volumes by 25–fold, yielding with a total assay volume

Page 3: Cyte Applications

of 2 µL with excellent Z’ factors and %CV’s.  

 

Figure 4. Compound screen using Plate 6 of LOPAC® (Sigma-RBI) performed in LV384 (panel A) and 1536 (panel B) well formats.  

Case Study – Deerac HTS Enables Miniaturized Kinase Assay Development in 1536-Well Plate FormatDiscoveRx relied upon the precision dispensing of the Deerac HTS to develop and miniaturize their ADP Hunter™ HS assay for kinase screening in 1536-well plates. This miniaturization enabled them to greatly increase assay throughput.

Figure 5. EC50 curves of PKA in 1, 2 and 4L reaction in 1536-well plates (left), and normalized (right).

Genomics

 

 

Page 4: Cyte Applications

Miniaturized Genomics Assays - Labcyte is ready when you are.

 

Increase throughput with 1536- and 384-well assays Contamination-free transfer of nucleic acid samples without tips Save cost by minimizing reagent consumption and waste

Labcyte Echo® and Deerac liquid handling systems provide high-speed, low-volume, contamination-free reagent dispensing and total assay assembly for genomics applications including PCR, genotyping, gene expression, DNA sequencing and RNAi. Together these powerful technologies provide better results and minimize consumption of plasticware and tips, delivering cost savings and reduced waste.

qPCR Reaction SetupFigure 1. The Labcyte Echo 555 liquid handler was used to prepare miniaturized quantitative PCR reactions in 2 µL and 5 µL total reaction volumes using just 10 – 500 nL of primers and probes. Results indicated highly consistent crossing point (Cp) values and low CVs. Full details here.

SNP Genotyping PCR and DNA Sequencing Automated plating of bacteria for colony picking RT-PCR and Gene Expression Analysis RNAi Assays

SNP Genotyping

 

Figure 2. SNP genotyping results using the Deerac GX liquid handler.

Case Study - Miniaturization and Reduction of Assay Costs KBiosciences (Hoddesdon, UK) perform SNP genotyping services in 384- and 1536–well plate formats. Using the Deerac GX liquid handler, they have miniaturized PCR reagent mix additions from 5 μL to 250 nL. This provides:

o Cost savings of up to 20-foldo Improvements in genotyping call-rate percentageso Reliable and robust performance over millions of assayso Increased sample throughput

For further information:A-100 Low Volume Pipetting in SNP Genotyping

Page 5: Cyte Applications

PCR and DNA SequencingLabcyte liquid handling systems are used in various stages of sample preparation and assay set-up for DNA sequencing:

PCR reaction set-up for DNA template preparation Rolling Circle Amplification (RCA) Sequence reaction set-up PCR reactions

 

 

 

Figure 3: Sequencing reactions prepared using the Deerac GX liquid handler. Independent channel control allows flexible sample layout and increases throughput and efficiency.

Automated plating of bacteria for colony pickingThe Deerac GX liquid handler enables the spotting of transformed bacteria in a defined and uniform pattern across an agar plate for optimum colony growth and increased colony picking efficiency.

 

 

 

 

For further information:A-110 DNA Sequencing at the Broad InstituteA-150 Automated Colony SpottingA-210 DNA Sequencing at the Gallo Research Center

RT-PCR and Gene Expression Analysis . 

Case Study:The Institute of Molecular Medicine at Trinity College Dublin used the Deerac HTS platform to reduce real-time PCR reaction volumes, yielding significant cost savings per reaction.

Page 6: Cyte Applications

 

Figure 4. PCR reactions at 2.5 L (left) and 1.25 L (right) prepared using the Deerac HTS liquid handler. Identical cycle times were obtained in for both volumes, and Cp precision was very low (0.2% CV at 2.5 L and 0.3% CV at 1.25 L). 

In gene expression analysis using qPCR, the Labcyte low-volume liquid handling systems enable high-precision and high-throughput dispensing of master/primer-probe mix and/or cDNA.

For further information please download:A-190 Miniaturizing Real Time PCR ReactionsA-200 Wash Procedures to Minimize cDNA Carryover 

RNAi AssaysHigh-throughput miniaturized siRNA library screening using Echo liquid handlers delivers more accurate signal at lower reaction volumes.   

Case Study: The Institute of Molecular Medicine at Trinity College Dublin used the Echo 555 liquid handler to prepare standard, reverse and lipid-free siRNA transfections. Transfections prepared using the Echo liquid handler yielded more representative inclusion intensities, even than those prepared using traditional manual pipetting.

For further information:

Miniaturized siRNA Transfection Using Acoustic Droplet Ejection 

High-Content Analysis

Labcyte non-contact liquid handling solutions deliver precise volumes of compounds and reagents to live cells for high-content analysis.

 

 

Page 7: Cyte Applications

Figure 5.Cells fixed and stained with the Deerac HTS liquid handler. Green = ERK, blue = nuclei. 

ArrayingLabcyte Echo liquid handlers, Deerac GX liquid handlers and Portrait spotters can be used to build low-density arrays on flat substrates. These instruments are compatible with a wide range of fluid types, enabling array fabrication using nucleic acids, proteins, chemicals, live cells and more.

Figure 6. Protein arrays spotted onto a flat surface in 96-well format using the Deerac GX liquid handler.

Imaging Mass SpectrometryWhat is MALDI Imaging Mass Spectrometry?

MALDI imaging mass spectrometry (IMS) links the universal detection capability of mass spectrometry with the positional information of molecular histology, generating mass spectra correlated to known locations within the tissue. MALDI-IMS can reveal the distribution of hundreds of ion signals ranging in size up to 20,000 Daltons. This information can be used to determine tumor margins, drug distribution throughout a tissue or organism, and proteomic profiles under different experimental or therapeutic conditions.

Protein profiles are obtained directly from frozen tissue sections. The Labcyte Portrait® 630 spotter deposits MALDI matrix, enzymes and other reagents in a user-defined pattern directly onto the tissue (Figure 1). The Portrait 630 user interface allows complete flexibility for the user to control reagent deposition parameters to optimize tissue penetration, reaction conditions, crystal formation, and positional resolution for different tissue types and applications.

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Figure 1. Left: H&E stained rat brain coronal section imaged on light microscope (10X). Right: Adjacent tissue section spotted with 25 mg/ml sinapinic acid in 1:1 acetonitrile/TFA (aqueous) using a Labcyte Portrait 630 spotter. (Tissue sections and microscopic image courtesy of Dr. Pierre Chaurand and Dr. Richard Caprioli, Vanderbilt University.)

The coordinates of each crystalline matrix spot are transferred to the MALDI mass spectrometer, and spectra are collected for each spot location (Figure 2.)

 

Figure 2: Sum spectrum of cumulative signal from all pixels at each m/z. Mass spectrometry was done using a Bruker Daltonics autoflex MALDI-TOF. Inset spectra are amplifications of the higher mass regions. (Spectrum courtesy of Dr. Pierre Chaurand and Dr. Richard Caprioli, Vanderbilt University.)

Molecules can be mapped directly to their locations on the tissue (Figure 3.)

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Figure 3. MALDI tissue maps constructed using BioMap image analysis software (www.maldi-msi.org). Each image represents the distribution of a single ion species in the tissue section. Left: m/z 4965 (thymosin beta-4); Middle: m/z 11306-11347 (histone H4); Right: m/z 12133 (cytochrome c). (Tissue maps courtesy of Dr. Pierre Chaurand and Dr. Richard Caprioli, Vanderbilt University.) 

Case Study - Portrait Spotter Enables On-Tissue Enzyme Digests The Portrait spotter accommodates a wide range of different reagents, including enzyme buffers and surfactants in addition to standard MALDI matrix solutions. Vanderbilt University used the Portrait spotter to deposit four different enzymes (trypsin, chymotrypsin, endoproteinase glu-C, and subtilisin) onto rat brain sections, followed by MALDI matrix deposited onto the same spot locations. In these experiments, each enzyme digest generated multiple unique peptide fragments, corresponding to myelin basic protein (Figure 4), tubulin, and synapsin.

Figure 4 – Sum spectra images of peptide fragments from myelin basic protein. Images were created using BioMap software (www.maldi-msi.org.)

Using MS/MS, peptide sequencing was performed to identify protein fragments directly from the tissue section. A peak at m/z=1603 in the chymotrypsin digest was identified with confidence as tubulin α-1 chain (Figure 5.)

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Other Applications Dispensing Cells and Beads Maintaining Sample Integrity Elimination of Serial Dilutions General Acoustic Droplet Ejection

Dispensing Cells and Beads

Automated Colony Spotting [applications note] A-180 Automated Dispensation of Yttrium Oxide SPA Imaging Beads [applications note] Acoustic Droplet Ejection and Laser Scanning Platforms Enable High-Throughput Cell Dispensing and

Quantitation S. Miller, J. Shieh, S. Pickett, Labcyte and Blueshift Technologies [poster] Automated Dispensation of Yttrium Oxide SPA Imaging Beads, into 1536-Well Plates using Deerac

Fluidics' Equator HTS Pipetting System [poster] High Content Screening with Sub-Wavelength Optical Imaging of Cells Transferred Acoustically into Sub-

Microliter Wells, A. Cabasug, M. Mutz, R. Stearns [poster]

Maintaining Sample Integrity

Applying the Labcyte MicroClime® Lid in Biological Assays to Improve Data Quality and Quantity, Adrian Freeman, M. Fagura, G. Bellfield & J. Unitt, AstraZeneca R&D Charnwood, UK, [poster]

Compound Preservation in Closed Loop Screening [poster] Hydration Free, Automation-Friendly Alternative for Compound Library Storage [poster] Optimum Conditions for Thermal Sealed Plates with Acoustic Liquid Transfers J. Shieh, M. Gomez, J.

Barco, I. Yates. [poster]

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Putting a Lid on Edge Effects: Protecting Compound Libraries with Lid-Based Environmental Control [poster]

DMSO Compound Library Volume Measurement and Hydration Monitoring in Storage Tubes and Microplates, presentation, LabFusion 2004 [Presentation]

Elimination of Serial Dilutions

Elimination of Serial Dilution [poster] Elimination of Serial Dilutions -Improving Throughput in High-Density Format Microplates While

Eliminating Cost and Improving Results [poster] New Tool for Automating Serial Dilutions for Activity ConfirmationExperiments, J. Cesarek and D. Nie,

AstraZeneca [poster]

General acoustic droplet ejection (ADE)

Acoustic Droplet Ejection "Touchless" Transfer This note describes acoustic droplet ejection (ADE) and it's background [applications note]

How Long Will A Survey Last When Performing Acoustic Droplet Ejections? [applications note] A Sound Idea by Laura Cutland, San Jose Business Journal, April 22, 2005 [Article] Acoustic droplet ejection in drug discovery, E Heron, R Ellson, J Olechno, Drug Plus International,

April/May 2006, pp 22-25 [Article] Acoustic Droplet Ejection Technology, J Olechno, R Stearns, R Ellson, E Heron, Innovations in

Pharmaceutical Technology, April, 2006, pp 40-43 [Article] Automating Compound Management Systems, Catherine Shaffer, Drug Discovery & Development, August

2005, pp 36-41 [Article] Continued Miniaturisation of Assay Technologies Drives Market for Nanolitre Dispensing, Drug Discovery

World, Summer 2004, pages = 43-54 Transfer of Low Nanoliter Volumes between Microwell Plates Using Focused Acoustics-Automation

Considerations, Journal of the Association of Laboratory Automation, 8 (5) 2003 pp34-39 [Article] Debunking the Myth: Validation of Fluorescein for Testing the Precision of Nanoliter Dispensing, D L

Harris and M Mutz, JALA, 11(4), 233-239, 2006 [Article] Improving IC50 Results with Acoustic Droplet Ejection, J Olechno, J Shieh, R Ellson, JALA, 11(4), 240-

246, 2006 In situ DMSO Hydration Measurements of HTS Compound Libraries, Ellson et al, Combinatorial

Chemistry & High Throughput Screening, 8(6), September 2005, pp 489-498 Click here to read the abstract of the paper [Article]

Labcyte Inc: Company Interview, The Wall Street Transcript, November 2003 [Article] Leaps in Dispensing Change Liquid Handling Landscape by Sean Keating, Managing Editor, Drug

Discovery & Development, January, 2005, 46-48 [Article] Marketing in Science and the Science of Marketing: Hugh Kennedy interviews Elaine Heron, CEO,

Labcyte, Hugh Kennedy, September 2007 [Article] Miniaturization and Low Volume Liquid Handling Take the Lead in Lab Automation, B Tulsi, Bioscience

Technology [Article] Nano to Next Gen: Automation Gets Personal, J Perkel, Science, Vol 319, Issue 5861, 345-347, January 18,

2008 Picoliter: Company Profile, American Laboratory, August 2003 [Article] Picoliter: enabling precise transfer of nanoliter and picoliter volumes, Drug Discovery Today 7 (5) (2002)

pp S32-S34 [Article] Precision Nanoliter Aqueous Transfer, J Shieh, S Pickett, J Olechno, GEN News, Vol 27 No 9, May 1 2007

[Article]

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Serial vs Direct Dilution Time to apply new thinking to IC50 determination and dose response analysis? J Comley (HTStec Inc), Drug Discovery World, Spring 2007, 36-50 [Article]

Sound Advice: Acoustic technologies facilitate ultra-low volume liquid handling, Today's Chemist at Work, October 2003 [Article]

Sound Effects American Laboratory, December 2004, pp 26-30 [Article] The Effects of Small Molecule and Protein Solutes on Acoustic Drop Ejection, J Shieh, R N Ellson and J

Olechno, JALA, 11(4), 227-232, 2006 [Article] Touchless Liquid Transfer for the Life Sciences Genetic Engineering News, November 15, 2004, 60-61

Rich Ellson interview by Nina Bell of Lab-on-a-chipcom, August 2004 [Article] Echo 550 (Click Here to Open) [Movie] Movie of acoustic droplet ejection into a well filled with aqueous buffer. This movie shows that when the

dense solution of DMSO is ejected into an inverted solution of buffer, the DMSO stays at the meniscus until the plate is returned to the normal position. The DMSO solution then diffuses gradually rather than as a bolus. [Movie]

Movie of pin tools adding DMSO to PBS solutions courtesy of Dr. Joerg Dreessen of Novartis. This movie shows the addition of DMSO (containing a dye for easy visualization) into a buffer using pin tools. The dense DMSO drops as an undiluted bolus to the bottom of the well. Cells adherent to the bottom of the cell are exposed to injuriously high levels of DMSO until the solution completely disperses. [Movie]

Stroboscopic movie of 2.5 nL droplets being ejected from a single well. This movie captures many individual droplets into one, easy to understand sequence. [Movie]

Best Practices for Assessing Nanoliter Transfer Performance for Labcyte Echo Liquid Handlers Using the Artel MVS [poster]

Debunking the Myth - Using Fluorescein in Analytical Measurements of Fluid Transfers [poster] Impact of Compound Concentration in Solutions on Acoustic Detection and Transfers [poster] Integrating Acoustic Droplet Ejection into the Laboratory Environment [poster] Keeping DMSO Concentration Below 0.5% to Minimize its Effect in HTS Assays [poster] [POSTER] Low Nanoliter Acoustic Transfer of Aqueous Fluids with High Precision and Accuracy Nanoliter Transfers in 1536-Well Format Using Acoustic Droplet Ejection [poster] Pharmacological Evaluation of Different Compound Dilution and Transfer Paradigms on an Enzyme Assay

in Low Volume 384-well Format, T. Spicer, Y. Fitzgerald, N. Burford, S. Matson, M. Chatterjee, M. Gilchrist, J. Myslik and J. O'Connell, Bristol-Myers Squibb [poster]

Testing the Precision of Acoustic Droplet Ejection - Measuring CVs for 5 Nanoliter Transfers [poster] The Microliter Assay Limbo [poster] Low and Sub Nanoliter Dispensing of Volatile Reagents for Sample Preparation and Crystallization,

presentation, SmallTalk 2003 [Presentation] Managing Compound Library Health Through Solvent Restoration, (7MB file) 8th International MipTec

Conference, May 2005 [Presentation] Nanoliter and Picoliter Liquid Handling for Life Science Applications, paper, Nanotech/Biotech

Convergence 2003 [Presentation] Non-Invasive Liquid and Live Cell Transfers for HTS, presentation, Lab Automation, 2003 [Presentation] High Density IC50 Analyses Using the Echo 550 Liquid Handler [Webinar]