To meet the growing demand for assays designed for use in automated cell imaging studies in microplate formats, Invitrogen (www.invitrogen.com) recently introduced the Cyquant NF cell proliferation assay kit. This new NF, or no freeze, version of the companys assay eliminates the freeze/thaw cell lysis step.
The main advantage of the Cyquant assay is its linear dynamic range of about 200-fold, from 50 cells to 10,000 cells per well, says Brett Williams, business segment director for imaging and microscopy at Molecular Probes, a business unit of Invitrogen. It improves workflow by allowing the combination of cell proliferation determination with other assays to interrogate multiple markers of cell health, adds Williams. The assay utilizes a new cell-permeant, DNA-binding dye together with a plasma membrane permeabilization reagent.
The trend now is to do cell-based assays in drug discovery, which are faster and more economical than animal studies, says Melissa Stolow, director of marketing for functional biology at Stratagene (www.stratagene.com). The companys Quantos cell proliferation assay lends itself to high-throughput screening in 96- or 6-well formats.
Cellomics (www.cellomics.com) portfolio of assays for HCS and imaging applications all yield cell count or cell density information as part of the assay output. When customers buy an assay for some other target, like apoptosis or transcription factor activation, they have the ability to look at proliferation or changes in cell count at the same time, says Sarah Burroughs Tencza, Ph.D., product manager for bioapplications at Cellomics. The company does not market a cell proliferation assay per se, but includes cell counta direct measure of cell number and cell proliferative activityas a core component of all of its HCS assays. For a more detailed assessment of what might be causing an increase or decrease in proliferation, Cellomics offers a cell viability assay, a cell cycle assay, and a mitotic index assay.
Cellomics reagents are validated for use with HCS, and now that the company is part of the Fisher Biosciences group, Judy Masucci, Ph.D., director of marketing and sales support at Cellomics, expects to see growth in the number of assay kits produced for HCS applications. Although users require only a fluorescence microscope to perform these assays, the ability to use them on a HCS platform allows for automation and multiplexing.
We are hearing from our end-users in pharma/biotech of a need for flexibility to scale up assays in terms of throughput for high-throughput screening applications, says Jeffrey Emch, product manager at Roche Applied Science (www.roche-applied science.com). In the future, we will see the market moving toward molecular markers for cell proliferation, as well as cell death and toxicity. To meet that emerging need, Roche recently introduced the LightCycler 480 platform for high-throughput, real-time PCR.
The most recent generation of ELISA BrdU assays are compatible with highthroughput screening formats. They offer good sensitivity down to 5,00010,000 cells/well and detection down to a 20% increase or decrease in DNA synthesis, says Michelle Moore, applications and technical consultant at Roche.
Cambrex (www.cambrex.com) ViaLight HS (high sensitivity) BioAssay is a one-step assay, scalable for high-throughput applications in 96- or 384-well formats. The assay is based on bioluminescent detection of cellular ATP and can measure down to 10 adherent or non-adherent mammalian cells per microwell. ViaLight has two key advantages; the ability to measure over a dynamic range of six decades and its long signal durationthe signal retains its strength and quality for six hours, according to D.G. Ferneyhough, segment manager for research products at Cambrex Biosciences Walkersville.
Promegas (www.promega.com) CellTiter-Glo Luminescent Cell Viability Assay measures ATP in a high-throughput micro-well format. ATP assays are capturing the market from tetrazolium assays, says Terry Riss, director of project management, R&D at Promega. They provide an immediate endpoint and minimize the risk of chemical interference, associated with tetrazolium or rosazurin reduction assays.
The CellTiter-Glo assay can detect 10 cells or fewer in a well and has been miniaturized for use in 1,536-well plates. According to Riss, it contains an engineered, highly stable form of luciferase, enabling the use of large amounts of detergent to stop any ATPase activity. The luminescent signal has a half-life typically greater than five hours. The use of luminescence eliminates problems of background noise associated with fluorescent assays that may be compromised by fluorescence interference from library compounds.