The escalating cost of drug discovery has driven the demand for fluorescence multiplexed cell-based assays. By exploiting the varying excitation/emission profiles of fluorophores to enable labeling of multiple targets in a single assay, researchers can maximize the data generated. Laser-scanning technologies are especially well suited to extending the utility of these content-rich multiplexed assays to primary screening campaigns due to the speed at which they can acquire and analyze data and the relatively small data files. This contrasts with imaging systems, which can be too slow to effectively run large primary screens and produce data too rich to interpret over large compound sets.
One fluorescence detector, the Acumen eX3 microplate cytometer, uses three scanning lasers (405 nm, 488 nm, 633 nm) to excite fluorescent objects on the bottom of microplates.1 Band pass and dichroic filters divide the resultant emissions into green, yellow, red, and far red wavelengths. Four photomultiplier tube detectors simultaneously monitor four colors per laser giving a maximum of 12 channels of data, which significantly extends the range of fluorescent reagents that can be combined in multicolor, multiplexed assays. High-content information is generated from the detected fluorescent intensities and displayed as three-dimensional profiles of objects. These profiles allow the calculation of a range of morphological and fluorescent parameters to define cell populations and biological responses. The instrument uses thresholding methods that enable “on-the-fly” cytometric analysis and dramatically reduces file sizes. Rapid plate read times enable high throughputs with up to 300,000 samples/day. With a large field of view (20 mm x 20 mm) whole-well scanning is standard, which overcomes the oft-seen problem of variable stimulation and random cell distribution; this enables normalization of biological responses to total cell number.
Multiplexed assays assays have been used in the simultaneous determination of the effect of cell cycle inhibitors on cell number, DNA content, cyclin B1 expression, and cytotoxicity.2 Multiplexed HCS assays are also invaluable in genome-wide RNAi screens. The Acumen eX3 is currently being used in an on-going genome-wide screen to determine the genomic instability, survival, and drug-resistance pathways in human cancer by the simultaneous measurement of changes in ploidy, cell survival, and apoptosis. Multiplexing is particularly advantageous when studying multi-step metabolic and signal-transduction pathways and cancer-specific multiple events where the measurement of multiple parameters are required.
While the performance of instruments with multiple lasers may currently exceed that of multi-color, multiplexed assay protocols, the capacity to further multiplex is only limited by reagents and will broaden as reagent developers release new products onto the market. Scientists are currently able to study 5 or 6 targets within a single experiment but as the range of commercially available dyes, antibody conjugates, and general fluorescent proteins increases, this could rise to as many as 10 or 12 targets per assay.
Paul Wylie is the Acumen product manager and has been working at TTP LabTech for 9 years. He received his PhD from Leicester University.
References
1. Bowen WP, Wylie PG. Application of laser-scanning fluorescence microplate cytometry in high content screening. Assay Drug Dev Technol. 2006;4(2):209-21.
2. Kriauciunas A, Roell W. A Perspective from Eli Lilly and Co, European Pharmaceutical Review, 11(3), 2006;42-51.
This article was published in Drug Discovery & Development magazine: Vol. 13, No. 1, January/February, 2010 pp. 22-23.
Filed Under: Drug Discovery
