High Throughput Automated High Content Screening using a BioCube™ System and Microplate Cytometry
Poster Nov 03, 2005
Wayne Bowen, Jason Quarles and Michael Catalano
AbstractMany high content analysis (HCA) platforms offer limited capability for primary screening due to their low throughput, large data files and incompatibility with high-density microplates, making it impractical to deploy such systems for high-throughput screening. Laser-scanning microplate cytometers, such as the Acumen Explorer, offer rapid read and analysis times (typically <10 minutes). In addition, 96, 384 and 1,536 well microplates can be analysed at equivalent plate read times since scanning is performed on an area and not well basis.
For example, in measuring protein kinase activation (a common high content assay), the multi-step nature of the protocol is complex and requires much coordinated labour to perform. Here, we model the integration of the Protedyne BioCube System and the Acumen Explorer for practical high-throughput, high content screening of protein kinases. Integration of cell culture, compound addition, immunodetection and analysis results in capacities up to 40,000 wells/day, with superior process control.
The scaling up of cell-based assays used for target identification and validation for primary screening campaigns is not a trivial exercise. Key factors include the culture of large numbers of cells, their plating into microplates, and the complexity of theassay protocol being applied. So-called high content assays are amongst the most demanding since they are involve multiplexing of fluorescent dyes, proteins or probes.
HCA is predominantly performed using CCD Imagers, but for high content screening (HCS) they may be excessively complex, offer limited throughput and create data storage issues. Laser-scanning microplate cytometers, such as the Acumen Explorer, combine the object-recognition capabilities of CCD Imagers with the fast read speeds of bulk fluorescence readers (typically <10minutes). In addition, 96, 384 and 1,536 well microplates can be analysed at equivalent plate read times since scanning is performed on an area and not well basis.
Protein kinases play a major regulatory role in many cellular pathways, such as those leading to proliferation, differentiation and apoptosis. Consequently, they are an important target for drug discovery research, especially oncology.Measurement of protein kinase activation in cells can achieved using specific antibodies requiring the application of a multi-step staining protocol.
Here, we model the integration of the Protedyne BioCube System and an Acumen Explorer for practical high-throughput, high content screening of protein kinases. Integration of cell culture, compound addition, immunodetection and analysis results in capacities up to 40,000 wells/day, with superior process control.
1. High Content Screening Using an Acumen Explorer Fluorescence Microplate Cytometer
• Fast plate read times (4 – 10 minutes)
• Scans 96, 384 and 1,536 plates in same time
• Small file sizes; down to kb in screening mode
• Multiplexing –up to 4 colours in a single read
• Whole well analysis
Acumen Explorer is a laser-scanning fluorescence microplate cytometer that offers high content screening at throughputs compatible with primary and secondary screening campaigns. The Acumen Explorer's precision optics deliver unparalled whole well scanning performance for the execution of multiplexed assays in drug discovery.
Key biological applications include determination of protein kinase activation, cell cycle analysis, and -following the introduction of Acumen Explorer 405 -beta-lactamase reporter gene analysis
2. Automation of High Content Screening on a BioCube™ System
The BioCube System is an advanced laboratory automation platform that brings industrial automation practices to the research laboratory. The BioCube System is based around an industrial-design 4-axis cartesian robot with modular, interchangeable SmartTools and accessories, and a flexible database-driven software architecture. This allows the system to be optimally configured for a variety of applications in addition to high content screening.
Key assay data can be supplied, and resultant information can be queried and retrieved, through files or a direct LIMS connection. This enables output data from the Acumen Explorer to be fed-back and utilized in a closed loop fashion for secondary sample hit picking or in-line real-time quality control.
3. High Content Analysis of Protein Kinase Activation Using Anti-Phosphokinase Antibodies
Activation of ERK through phosphorylation by its upstream activator MEK in CHO cells. FCS-activated ERK was detected using phospho-p44/42 MAP kinase antibody (CST #9101) and goat anti-rabbit FITC antibody (Jackson; 111-096-045). Cells were counter-stained with propidium iodide. Well Views from the Acumen Explorer software show control well (left) and FCS-treated well (right). The ability of Acumen Explorer to analyse the entire well permits normalisation of responses to the total cell number, and compensate for variable cell growth or kinase stimulation within the well.
4. Automated Protein Kinase Screening using a BioCube System and an Acumen Explorer
Estimated Performance (per 24 hours)
High Content Screening Configurations
|TTP LabTech Ltd, Melbourn Science Park, Royston, Hertfordshire, SG8 6EE, UK. Phone +44 1763 262626 Visit us at www.ttplabtech.com/explorer|
|Protedyne Corporation, 1000 Day Hill Road, Windsor, Connecticut, 06095, USA. Phone +1 (860) 683-1860 Visit us at www.protedyne.com|