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The 8200 Cellular Detection System (Fluorescent Microvolume Assay Technology – FMAT) from Applied Biosystems is a laser scanning macro-confocal fluorescent plate reader.
FMAT scans the central 1mm2 area of a micro well to a depth of 100μm from the base of the well using a red 633nm Helium-Neon laser to identify “objects” within the focal plane. Objects such as cells or beads and associated fluorescence are detected and measured by two photomultiplier tubes (PMT1, 650-685 nm; PMT2, 685-720 nm) simultaneously. Fluorescence not associated with objects is undetected, so that the signal-to-noise ratio is dramatically increased. In addition, a 633nm excitation source reduces interference from the auto-fluorescence of compounds and plastic.
These features enable FMAT to perform homogeneous assays without the need to remove unbound label using time consuming washing steps that introduce significant assay variability.
Cell-based assays may be conducted on live or fixed cells with both adherent and suspension cell lines. Bead-based assays are also easy to perform and existing ELISAs are readily converted to homogeneous FMAT formats (FLISA) by coating beads with capture antibodies (Ref – J Biomol Screen March 2008 vol. 13 no. 3 210-217).
Examples of assays and applications include:
We have extensive experience of developing whole cell binding assays for chemokine receptors. Chemokine ligands can be labelled with Alexa-647 for the measurement of receptor binding. Competition assays can be set up between labelled chemokine and compounds for screening purposes. These assays are robust and have been used for high throughput screening and hit to lead campaigns.
Comparison of labelled versus control
HEK-293 cells stably transfected with a chemokine receptor were treated with Alexa-labelled chemokine ligand (left panel) or chemokine in the presence of an excess of competing compound (right panel). Cellular fluorescence (left) is consistent with ligand binding to receptors and can be quantitated using the FMAT. In the presence of competing ligands or compounds this staining was diminished or completely absent (right).
The appearance of phosphatidylserine (PS) in the outer surface of the plasma membrane is an early marker of apoptosis. Labelling of the nuclei of live cells with CentriRed and labelling of phosphatidyl serine in the plasma membrane with Alexa-647 labelled Annexin V allows quantitation of the number of apoptotic cells.
The FMAT can be used to monitor CD marker expression using fluorescently tagged antibodies. An example of an assay we have used is the expression of CD11b on inflammatory cells detected with Alexa-647 labelled anti- CD11b.
Screening of hybridoma supernatants using the FMAT is straightforward with no interference from the media and can be performed in a homogeneous format without the need for wash steps. The format allows use of adherent and suspension cells expressing surface target proteins.