FLIPRS Propel Labs Along

Molecular Devices' FLIPR384 System The accelerating pace of drug discovery has spawned an increasing need for functional assays using living cells. Automating these assays for high-throughput systems, however, has proven to be difficult. Noting that the most common biological assays employed in high-throughput settings rely on some kind of fluorescent measurement, Molecular Devices Corporation of Sunnyvale, Calif., has developed an automated solution for functional whole-cell screening. Launched in 1996, the Fluorometric Imaging Plate Reader, otherwise known as the FLIPR, is a unique combination of optics, automated pipetting, and temperature control. Designed to perform high-throughput screening assays using adherent and nonadherent cells, the FLIPR is ideally suited for kinetic, cell-based assays such as measuring changes in intracellular calcium levels and membrane potential. According to Simon Pitchford, product marketing manager for cell analysis at Molecular Devices, the FLIPR technology has been well accepted by the industry. "The FLIPR was designed to fulfill the needs of the customer, rather than being technology forced on the marketplace," he said in a telephone interview. So how does the FLIPR work? FLIPR integrates a powerful argon laser, a CCD (charged couple device) imaging camera, a patented optical detection scheme, and a programmable 96-well pipettor to perform fluorometric analyses on all 96 wells of a microplate simultaneously. The system generates real-time kinetic data by stimulating and reading all 96 wells in 1-second intervals. Typically, the argon laser excites a suitable fluorescent dye. The resultant emitted light is detected by a cooled CCD camera that acts as an integrated detector, accumulating signal over the duration of the exposure. Sensitivity is further enhanced by the FLIPR's optical scheme, which limits the depth of field of the CCD camera to a few hundred microns on the bottom of each well, or essentially at the level of the cell monolayer. This technique reduces the background fluorescence from extracellular dye by about an order of magnitude. Taken together, these systems combine into an extremely sensitive fluorescence detector. Of course, the FLIPR needs the ability to stimulate all 96 wells of a microplate simultaneously to be a useful screening tool. To accomplish this, it possesses an integrated, programmable 96-well pipettor. Handling a volume range of 5 to 250 µl with a 1 percent accuracy and precision of 20 µl, the pipettor uses disposable tips to transfer compounds from three reagent plates onto a single assay plate. And because fluorescent dyes exhibit temperature sensitivities that can impact an assay, the incubation chamber on the FLIPR holds its temperature to within 0.1°C over a 30-minute assay period. Recognizing the shift to higher throughput and reduced assay volumes, Molecular Devices has designed another FLIPR system capable of handling both 96-well and 384-well plates. Incorporating much of the same technology as the FLIPR, the FLIPR384 is also equipped with interchangeable 96- and 384-channel pipetting heads and includes advanced automation capabilities. Specifically, the FLIPR384 operates with an optional integrated plate stacker and a specialized stage that shuttles up to 50 cell and reagent plates through the system. This mode also features an integrated pipet tip washer. Alternatively, the FLIPR384 integrates into linear robotic systems that use robotic arms for manipulating plates and supplies. As stated by the product literature from Molecular Devices, the move to 384-well plates translates into significant reductions in reagent usage. Whereas a well on a 96-well plate needs 50 to 100 µl of fluid additions, a well on a 384-well plate needs only 2 to 30 µl. Overall, savings from decreased volumes and greater throughput result in an estimated working cost of 15 cents per well for the FLIPR384 compared to 40 cents per well for the FLIPR. Daily plate throughput rates for the FLIPRs reach 150 plates and higher, so in a typical 8-hour shift, 11,520 to 57,600 wells are processed with a minimum amount of operator intervention. Drug discovery time can be reduced by using this single-assay platform to run both the screening and the lead optimization functions. The strength of the FLIPR instruments is their ability to capture real-time kinetic data, enabling the identification of a potential hit within seconds of its addition to an assay. Indeed, one of the key features of FLIPR technology is its data fidelity, allowing researchers to monitor single-well determinations. The software also performs data analysis and reduction and can export data to spreadsheet programs. Roche Bioscience of Palo Alto, Calif., owns a FLIPR and a FLIPR384, which it uses for high-throughput screening assays that measure cellular calcium levels. Wilhelm Lachnit, research scientist at Roche Bioscience, reports that a typical run processes 90 to 120 96-well plates in an 8-hour period. "I am absolutely satisfied with the performance of the FLIPR. I was pleasantly surprised at how reproducible the results are," he said. Lachnit also reports that in a typical week, 80% of the usage of the FLIPR involves high-throughput screening applications, with quantitative pharmacological studies largely taking up the remainder of the instrument's use. The FLIPR and the FLIPR384 are adaptable to just about any functional cell-based assay that incorporates a single-wavelength fluorescent dye with visible wavelength excitation. Method development on these instruments is facilitated by the speed at which they gather data, their abilities to make dual-emission measurements, and flexible software. Assays such as membrane potential changes and intracellular calcium level measurements, the most common application for the FLIPRs, have been well documented in product literature, FLIPR application notes, and the scientific literature as well. The author, Michael Brush, can be contacted at mdbrush@compuserve.com. For more information, contact Molecular Devices Corporation at (800) 635-5577 or visit the company Web site at www.moleculardevices.com.

FLIPRS Propel Labs Along

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