A flow cytometer equipped with a 488- or 532-nm laser and a 635-nm laser capable of detecting and distinguishing fluorescence emission at 576 and 670 nm (off the 488-nm laser) and 660 nm (off the 635-nm laser) such as a FACSCalibur or 576 nm (off the 532-nm laser) and 660 and >680 nm (off the 635-nm laser) such as a FACSArray bioanalyzer is needed to perform CBA Flex Set bead assays. For assays run on the FACSCalibur 12 X 75-mm sample acquisition tubes are needed. FACSArray tests are run on microtiter plates, such as Millipore MultiScreen® BV 1.2-pm clear nonsterile filter plates. Plate washing is performed on a Millipore MultiScreen vacuum manifold, the plates are mixed on a digital microplate stirrer. Data acquisition and analysis requires availability of CellQuest, FACSComp, and FCAP Array software packages.
The cell signaling Flex Set assays are two-site sandwich multiplex immuno-assays, which are available as total protein or phophorylated protein assays (Table 2). The assays are using cell lysates as samples and provide quantitative results based on standard curves constructed with recombinant protein standards. The sample data are analyzed using the FCAP Array software and the results are expressed as arbitrary U/mL for each analyte.
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