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April 15, 2011 (Vol. 31, No. 8)

High-Throughput Flow Cytometry

Speeding Up the Process Has Increased the Technology's Utility in Systems Biology

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    Figure 3. The final result for this assay is a direct IC50 readout from listmode files. This figure will be generated if the “plot” icon is clicked on Figure 2. Drug concentrations are shown on the x axis.

    In the next step, a process icon such as a Boolean operator is identified, as well as a “container” that represents a layout to define the drug dilutions of interest. Finally, a plotting icon can be used to plot multiple containers on one graph. The final result is an IC50 curve and reported value as shown in Figure 3. IC50 plots are generated both visually and as XL spreadsheets that can be uploaded to any system. In fact, any result from any component can be visualized or delivered via spreadsheet files.

    The process of creating an automated flow-cytometry screen is a comprehensive one. It requires appropriate design of the assay systems, creation of automated plate preparation, robotically managed sample collection, and, finally, a fast, very specific result-generation system. Traditional flow-cytometry analysis approaches are highly unlikely to be successful in dealing with the superabundance of data produced by HT systems.

    Further, the concept of sequential analysis routinely used in flow-cytometry analysis simply fails when faced with the demand of large plate formats. This opens an opportunity to create relatively specific analytical tools that are designed to focus on multiple-well plates and to operate in a way that is fast and efficient.

    So why use flow cytometry? A great majority of HT screening is performed on image-based instruments. This has become the standard across pharmaceutical companies and academia. However, it is time to rethink this logic. Flow cytometry has many advantages, particularly with suspended cells such as the HL60 cells used in the aforementioned assays. However, even attached cells can be successfully lifted from plates for flow cytometry.

    A significant advantage of flow cytometry is that systems where multiple populations might exist, either physically or functionally, can be successfully managed, whereas imaging has great difficulty. Sample speed is generally much faster in flow cytometry than in imaging. A typical flow cytometry 384-well three- or four-color assay with 5,000 cells per well can easily be run in 10 minutes. It would take no longer to collect eight or nine fluorescent colors.

    Collecting these numbers of cells and variables by imaging is well beyond even the fastest systems. No special plates are required for flow. The quality of the plate directly impacts the results in imaging, where minor variations in flatness or transmission can dramatically alter the results.

    In flow, every cell is independently analyzed in the absence of all other cells. Imaging requires complex segmentation algorithms to separate cells and frequently the result is a compromise in identification. It is true that flow cytometry cannot provide the same type of morphological or location-specific data that imaging systems can, but the advantages flow cytometry brings for other analytical processes is very significant. High-speed flow cytometers matched with robotic delivery systems and robotic preparative systems are now an attractive alternative to current high-throughput microscopy-based systems. Speeding the analysis process has made high-throughput flow cytometry a transformational tool in the systems biology chest.

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