Multiplexing cell-based assays

Fluorescent microvolume assay technology (FMAT ) is a bead-based or cell-based fluorescent technology for homogeneous ELISA-like assays. In FMAT, a laser beam is focused on the bottom of the assay well and the localised fluorescence intensity bound to beads (or cells) is detected as an area of intense fluorescence over the unbound and background fluorescence in solution. Different analytes can be detected with appropriate fluorophores and, by using different sized beads, the assay can be multiplexed to monitor multiple analytes.13... 

Another important set of multiplexed assays monitor mRNA transcript levels. The expression level of all the genes involved in a known signal transduction pathway or other selective genes can be monitored simultaneously as a way of following compound effects on a cell. The current technologies for multiple mRNA detection include quantitative reverse transcriptional PCR (qRT-PCR), qNPA (quantitative nuclease protection assays), mass array assay technologies and branched DNA detection on Luminex beads (Panomics). The applications of such multiplexed in vitro and cell-based detection systems should provide more predicative information in hit finding and lead characterisation. 

Cell-Based Bioassay For a cell-based Nab bioassay, treated cells respond directly or indirectly to the drug in a concentration-dependent manner. Possible biological responses of the cells to chug treatment include cell proliferation, apoptosis, phosphorylation, chemokine release, and expression of proteins or genes [15,22,28 30]. These responses may by quantitated by techniques such as immunoassay, multiplex assays [31], flow cytometry [23], and gene expression profiling [32]. 

Bogojevic D, Chamberlain MD, Barbulovic-Nad I, Wheeler AR (2012) A digital microfluidic method for multiplexed cell-based apoptosis assays. Lab Chip 12(3) 627-634... 

D.A., and Imperiali, B. (2005) A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell 23. lysates. Nat, Methods, 2, 277 -284. 

The other approach is based largely on informatics. In such an approach, tumors would be profiled in contrast to normal tissue. Tumor-specific markers would be identified via microarrays, as has been done in many publications already. From here, the list of tumor-specific markers would be analyzed to determine if any of these markers represented proteins which were likely to be secreted out of the cell and which may be detected in the peripheral blood stream. Preferably multiple markers would be identified that could be tested using multiplex ELISA assays (antibody arrays). Such work will take time, however, because once the potential markers are identified, antibodies must be generated, validated, and tested for effectiveness as an early diagnostic tool. Such work is being done, but little has been published so far. 

A major advantage of this method is the ability to multiplex with other assays. The GF-AFC substrate used to detect viable cells was designed for use in combination with another substrate that selectively detects protease activity from dead cells (Niles, Moravec, and Riss 2008). The method used to measure dead cells is based on the /riv-Ala-Ala-Phe-rhodaminc 110 (AAF-R110) protease substrate. This substrate is non-permeable thus viable cells do not substantially contribute to signal. Dead cells with compromised membranes leak protease activity into the surrounding medium... 

HCS instruments take microscope-based images of a limited region of each microtitre plate well with sub-cellular resolution. Proteins of interest can be detected by a fluorescent tag, such as GFP, or are identified by fluorescent antibodies. One of the characteristics of HCS assays is the ability to not only monitor the levels of different proteins within the cells but also their location, the morphology and shape of the cells by multiplexing several labels.90 92... 

Interesting studies were also performed based on a suspension of beads in conjunction with flow cytometry measurements [23,24], Flow cytometry, which was the standard methodology for cell population study during the last 20 years, has now begun to serve for in vitro microspheres analysis [25]. Such systems were described as multiplex microsphere bead assays and were used to detect different nucleic acid sequences hybridized on beads having different properties (size, fluorescent label). 

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