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Title and Authors Year Type

297 Distinct Cell Lines: A High-Content Analysis Assay and a Full-Automation Design Solely Using Noncontact Liquid Dispensing

Rodriguez R, Alfonso J, O'Day C, Ovechkina Y, Ward C

Institution(s): MDS PharmaServices, Inc.
Related Applications: HTS and Secondary Screening
Related Product(s): Echo® Liquid Handler for Screening, Echo® Liquid Handler for Screening and OMICS

This review assesses the quality of the data acquired over a 13-week period from a High-Content Analysis screening project that used 297 unique cell lines. This article also evaluates the proficiency of a “tipless” (i.e., does not use disposable tips) full-automation design used for this project that prioritizes intralab system mobility and system configuration mutability. The request to assay a large number of cell lines with poorly characterized growth rates led us to devise an MDS PharmaServices, Inc. proprietary algorithm in an effort to select the proper cell plating density for each cell line. The performance metrics include coefficients of variation (CVs) of Controls for the cell plating data and Data Set Mean CVs for assessing replicate propinquity (i.e., how close the replicates are to each other). The performance of the automation system and our algorithm for this project produced data of superior quality.

HeLa, automation,  SK-MEL-28, bolus, high-content screening, cross-contamination, cost reduction, INCell, cell based assays

Journal of Laboratory Automation Oct 2007 vol. 12 no. 5 318-326, 10.1016/j.jala.2007.06.004
 

Link: http://jla.sagepub.com/content/12/5/318.full
2007 Article

300 Nanoliter qPCR in a One-Step Process from Cells to Cp Utilizing the Echo® Liquid Handler

Glazer C, Lee H, Datwani S, Sonntag M

Institution(s): Labcyte Inc.
Related Applications: Gene Expression
Related Product(s): Echo® Liquid Handler for Screening and OMICS, Echo® Liquid Handler for OMICS

Recent innovations in cell lysis reagents and high throughput quantitative PCR miniaturization have allowed researchers to increase throughput while decreasing labor and reagent costs. To ensure high quality data, the liquid handling employed in such low-volume reactions must be precise and accurate. This study utilized the Echo 555 liquid handler (Labcyte Inc., Sunnyvale, CA), the Real-time ready Cell Lysis kit and the LightCycler® 480 system (Roche Applied Science).  Cells were grown in tissue culture plates, lysed with the Real-time Lysis solution and then transferred in nanoliter quantities using the Echo liquid handler into 384-well qPCR plates.  The plates were then cycled in the LightCycler and the resulting amplification analyzed.  Results show excellent standard deviations of less than 0.5 and coefficient of variations of less than 1.5% with volumes as low as 300 nanoliters. The results for this application illustrates the new capabilities of the Echo liquid handler to enable assay setup and reduce reaction volumes resulting in reagent and consumables savings with higher throughput.

quantitative PCR, real-time PCR, rt-PCR, cost reduction, SBS 2011
 

Link: None
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2011 Poster

3D Imaging by Mass Spectrometry: A New Frontier

Seeley E H, Caprioli R M

Institution(s): Vanderbilt University
Related Applications:
Related Product(s): Portrait® 630 Spotter

Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here, we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.

Anal Chem. 2012 Mar 6;84(5):2105-10. doi: 10.1021/ac2032707.

Link: http://pubs.acs.org/doi/pdf/10.1021/ac2032707
2012 Article

A Bioluminogenic HDAC Activity Assay: Validation and Screening

Halley F, Reinshagen J, Ellinger B, Wolf M, Niles A L, Evans N J, Kirkland T A, Wagner J M, Jung M, Gribbon P, Gul S

Institution(s): European ScreeningPort GmbH, Promega Corporation, Promega Biosciences, Institute of Pharmaceutical Sciences Albert-Ludwigs-Universitat Freiburg
Related Applications: HTS and Secondary Screening
Related Product(s): Echo® Liquid Handler for Screening, Echo® Liquid Handler for Screening and OMICS

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z′), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

J Biomol Screen December 2011 vol. 16 no. 10 1227-1235

Link: http://jbx.sagepub.com/content/16/10/1227.abstract
2011 Article

A Cell-Based High-Throughput Screening Assay to Measure Cellular Histone H3 Lys27 Trimethylation with a Modified Dissociation-Enhanced Lanthanide Fluorescent Immunoassay

Xie W, Ames R S, Li H

Institution(s): GlaxoSmithKline
Related Applications: HTS and Secondary Screening
Related Product(s): Echo® Liquid Handler for Screening, Echo® Liquid Handler for Screening and OMICS

Histone proteins are subject to several modifications, including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These posttranslational modifications play critical roles in chromatin structure and gene transcription. Because of their involvement in the progression of a variety of diseases, histone modifications are attracting increased attention. We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of “freeze-thaw” and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for high-throughput screening. The findings presented here should be applicable to other assays involving binding and extraction of histone proteins.

J Biomol Screen January 2012 vol. 17 no. 1 99-107

Link: http://jbx.sagepub.com/content/17/1/99.abstract
2012 Article

A Chemical Genomics Screen to Discover Genes That Modulate Neural Stem Cell Differentiation

Kim K J, Wang J, Xu X, Wu S, Zhang W, Qin Z, Wu F, Liu A, Zhao Y, Fang H, Zhu M, Zhao J, Zhong Z

Institution(s): GlaxoSmithKline
Related Applications: HTS and Secondary Screening, Sample Management
Related Product(s): Echo® Liquid Handler for Screening, Echo® Liquid Handler for Screening and OMICS

The authors designed a chemical genomics screen with the aim of understanding genes and pathways that modulate neural stem/precursor cell differentiation. Multipotent mouse neural precursor cells isolated from cortices of embryonic day 12 (E12) embryos were subjected to spontaneous differentiation triggered by growth factor withdrawal. A quantitative whole-well immunofluorescence assay was set up to screen tool compound sets to identify small molecules with potent, dose-dependent, and reproducible effects on increasing neural stem cell differentiation toward neuronal lineage. Among the pro-neuronal compounds, kinase inhibitors were shown to exert pro-neuronal effect via a signaling pathway associated with the kinase. The global effect of hit compounds on modulating neuronal differentiation was confirmed by an in vivo mouse study and human neural stem cells culture. This study demonstrates that a phenotypic assay using cell type–specific antibody markers can be used for a large-scale compound screen to discover targets and pathways with impacts on differentiation of lineage-restricted precursor cells toward specific lineages.

J Biomol Screen February 2012 vol. 17 no. 2 129-139

Link: http://jbx.sagepub.com/content/17/2/129.abstract
2012 Article

A High-Content Glucocorticoid Receptor Translocation Assay for Compound Mechanism-of-Action Evaluation

Agler M, Prack M, Zhu Y, Kolb J, Nowak K, Ryseck R, Shen D, Cvijic M, Somerville J, Nadler S, Chen T

Institution(s): Bristol-Myers Squibb, St. Jude Children's Hospital
Related Applications: HTS and Secondary Screening, Sample Management
Related Product(s): Echo® Liquid Handler for Screening, Echo® Liquid Handler for Screening and OMICS

Ligand-induced cytoplasm to nucleus translocation is a critical event in the nuclear receptor (NR) signal transduction cascade. The development of green fluorescent proteins and their color variants fused with NRs, along with the recent developments in automated cellular imaging technologies, has provided unique tools to monitor and quantify the NR translocation events. These technology developments have important implications in the mechanistic evaluation of NR signaling and provide a powerful tool for drug discovery. The unique challenges for developing a robust NR translocation assay include cytotoxicity accompanied with chronic overexpression of NRs, basal translocation induced by serum present in culture medium, and interference from endogenous NRs, as well as subcellular dynamics. The authors have developed a robust assay system for the glucocorticoid receptor (GR) that was applied to a panel of nuclear receptor ligands. Using a high-content imaging system, ligand-induced, dose-dependent GR nuclear translocation was quantified and a correlation with other conventional assays established.

glucocorticoid receptor, nuclear translocation, high content screening, green fluorescent proteins, androgen receptor, mechanism of action

J Biomol Screen December 207 vol. 12 no. 8 1029-1041,

 

Link: http://jbx.sagepub.com/content/12/8/1029.full.pdf+html
2007 Article

A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae

Rasmussen L, White E L, Pathak A,  Ayala J C,Wang H, Wu J H, Benitez J, Silva A J

Institution(s): Southern Research Institute, Morehouse School of Medicine
Related Applications: Biochemical Assays, HTS and Secondary Screening
Related Product(s): Echo® Liquid Handler for OMICS , Echo® Liquid Handler for Screening and OMICS

Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot) phenotype and is specific for the Na+-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na+ bioenergetics, such as the function of the primary Na+ pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na+-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

Antimicrob Agents Chemother. 2011 September; 55(9): 4134–4143. doi:  10.1128/AAC.00482-11

Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165335/
2011 Article

A Miniaturized and Automated P450-Glo™ Screening System Using the Echo® Liquid Handler

Wang J, Barco J, Worzella T, Kershner K

Institution(s): Labcyte Inc., Promega
Related Applications: ADME Tox
Related Product(s): Echo® Liquid Handler for Screening and OMICS, Echo® Liquid Handler for OMICS

Early assessment of cytochrome P450 (CYP) inhibition has become an essential component of drug discovery screening. The CYP3A4 enzyme is the most abundant enzyme in the liver and plays a major role in metabolizing many xenobiotics in humans. The Promega P450-Glo™ CYP3A4 Assay (Luciferin-IPA) offers an extremely sensitive, high throughput and specific luminescence assay for the examination of CYP3A4 inhibition with pooled Human Liver Microsomes.  The Echo 555 liquid handler provides precise and accurate acoustic transfer free of cross-contamination risk.  Here, we demonstrate optimization of the P450-Glo™ Assay with Human Liver Microsomes (HLM) in low volume format using the Echo 555 liquid handler.  We show that the Echo 555 liquid handler can transfer microsomes, inhibitors and substrate in nanoliter volumes, providing a sensitive and robust small scale assay.  IC50 results for four known CYP3A4 inhibitors using our miniaturized assay compared favorably to previously reported values in the literature.

Link: None
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2012 Poster

A New Approach to High throughput ADME Assay Screening

Wang J, Segre G and Wu S

Institution(s): Labcyte Inc., Quintara Discovery
Related Applications: ADME Tox
Related Product(s): Echo® 525 Liquid Handler

Early ADMET assessment is expected to not only improve the overall quality of drug candidates, but also shorten the drug discovery and development process. Time-dependent CYP450 inhibition and metabolic stability assays are critical in identifying drug candidates that may have undesirable drug-drug interactions or sub-optimal pharmacokinetic properties. The cost of profiling these assays during early drug development with large numbers of compounds however, can dramatically increase the expense of drug discovery. One objective for pharmaceutical companies is to find less expensive, more reliable and higher throughput ADMET methods that can be moved upstream. Here, we present a miniaturization method using the Echo® liquid handler to evaluate ADMET characteristics. The Echo liquid handler uses non-contact acoustic energy to transfer low volumes compounds and reagents. This new method not only generates high quality and reliable IC50 and intrinsic clearance values, but also significantly reduces reagent costs.

Link: None
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2013 Poster