High Throughput and High Content Screening Using Peptides

2005 ◽  
pp. 347-384
Author(s):  
Paul Hamilton ◽  
Robert Carlson ◽  
Robin Hyde-Deruyscher
Keyword(s):  
High Throughput ◽  
High Content Screening

scholarly journals A Novel Cell-Based, High-Content Assay for Phosphorylation of Lats2 by Aurora A

2011 ◽  
Vol 16 (8) ◽  
pp. 925-931 ◽  
Author(s):  
Amy Emery ◽  
David A. Sorrell ◽  
Stacy Lawrence ◽  
Emma Easthope ◽  
Mark Stockdale ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
High Content Screening ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Assay Performance ◽  
A Cell ◽  
Significant Addition ◽  
Specific Inhibitors

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration–response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.

scholarly journals HighVia—A Flexible Live-Cell High-Content Screening Pipeline to Assess Cellular Toxicity

2020 ◽  
Vol 25 (7) ◽  
pp. 801-811 ◽  
Author(s):  
Alison Howarth ◽  
Martin Schröder ◽  
Raquel C. Montenegro ◽  
David H. Drewry ◽  
Heba Sailem ◽  
...  
Keyword(s):  
Cell Viability ◽  
High Throughput ◽  
Early Stage ◽  
Accurate Determination ◽  
Live Cell ◽  
Fluorescent Imaging ◽  
High Content Screening ◽  
Imaging Protocol ◽  
Target Discovery ◽  
Oncology Research

High-content screening to monitor disease-modifying phenotypes upon small-molecule addition has become an essential component of many drug and target discovery platforms. One of the most common phenotypic approaches, especially in the field of oncology research, is the assessment of cell viability. However, frequently used viability readouts employing metabolic proxy assays based on homogeneous colorimetric/fluorescent reagents are one-dimensional, provide limited information, and can in many cases yield conflicting or difficult-to-interpret results, leading to misinterpretation of data and wasted resources.The resurgence of high-content, phenotypic screening has significantly improved the quality and breadth of cell viability data, which can be obtained at the very earliest stages of drug and target discovery. Here, we describe a relatively inexpensive, high-throughput, high-content, multiparametric, fluorescent imaging protocol using a live-cell method of three fluorescent probes (Hoechst, Yo-Pro-3, and annexin V), that is amenable to the addition of further fluorophores. The protocol enables the accurate description and profiling of multiple cell death mechanisms, including apoptosis and necrosis, as well as accurate determination of compound IC50, and has been validated on a range of high-content imagers and image analysis software. To validate the protocol, we have used a small library of approximately 200 narrow-spectrum kinase inhibitors and clinically approved drugs. This fully developed, easy-to-use pipeline has subsequently been implemented in several academic screening facilities, yielding fast, flexible, and rich cell viability data for a range of early-stage high-throughput drug and target discovery programs.

Challenges and Opportunities in Enabling High-Throughput, Miniaturized High Content Screening

2017 ◽  
pp. 165-191 ◽  
Author(s):  
Debra Nickischer ◽  
Lisa Elkin ◽  
Normand Cloutier ◽  
Jonathan O’Connell ◽  
Martyn Banks ◽  
...  
Keyword(s):  
High Throughput ◽  
High Content Screening ◽  
Challenges And Opportunities

scholarly journals Automated Analytical Measurement Processes Using a Dual-Arm Robotic System

2019 ◽  
Vol 24 (3) ◽  
pp. 354-356
Author(s):  
Heidi Fleischer ◽  
Shalaka Joshi ◽  
Thomas Roddelkopf ◽  
Michael Klos ◽  
Kerstin Thurow
Keyword(s):  
High Throughput ◽  
High Content Screening ◽  
Automation System ◽  
Analytical Measurement ◽  
Flexible Robots ◽  
Liquid Handling ◽  
Human Operators ◽  
Dual Arm Robot ◽  
Dual Arm ◽  
Direct Sample

The demand for automation in the analytical laboratory is high. In contrast to well-automated bioscreening and high-throughput and high-content screening processes, analytical measurement procedures are complex in their structure and changing frequently. Not only do robotic units have to perform transportation or specific single tasks, but also flexible robots are needed to cover several tasks, including transportation and direct sample manipulation. Due to their human-like structure, dual-arm robots are predestined for analytical measurement processes. A new study published in the journal Energies presents a novel integration of electronic piston pipettes into an automation system using a dual-arm robot to perform liquid handling tasks similar to human operators. In this commentary, the main findings are highlighted and discussed.

scholarly journals Apoptosis assessment in high-content and high-throughput screening assays

BioTechniques ◽  
2021 ◽  
Vol 70 (6) ◽  
pp. 309-318
Author(s):  
Céline Rens ◽  
Tirosh Shapira ◽  
Sandra Peña-Diaz ◽  
Joseph D Chao ◽  
Tom Pfeifer ◽  
...  
Keyword(s):  
High Throughput ◽  
Gold Standard ◽  
High Throughput Screening ◽  
High Content Screening ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hoechst 33342 ◽  
Monitoring Method ◽  
Multiple Cell ◽  
Screening Assays ◽  
Intracellular Infections

Here the authors describe the development of AUTOptosis, an economical and rapid apoptosis monitoring method suitable for high-content and high-throughput screening assays. AUTOptosis is based on the quantification of nuclei intensity via staining with Hoechst 33342. First, the authors calibrated the method using standard apoptosis inducers in multiple cell lines. Next, the authors validated the applicability of this approach to high-content screening using a small library of compounds and compared it with the terminal deoxynucleotidyl transferase dUTP nick end labeling gold standard. Finally, the authors demonstrated the specificity of the method by using AUTOposis to detect apoptosis triggered by Mycobacterium tuberculosis intracellular infections.

High-content screening of small compounds on human embryonic stem cells

2010 ◽  
Vol 38 (4) ◽  
pp. 1046-1050 ◽  
Author(s):  
Ivana Barbaric ◽  
Paul J. Gokhale ◽  
Peter W. Andrews
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Cell Biology ◽  
Es Cells ◽  
Embryonic Stem ◽  
High Content Screening ◽  
Cellular Models ◽  
Compound Libraries

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

Sign in / Sign up

Export Citation Format

Share Document