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Abstract for Multiplexing Cell-Based Assays Using Standard
Luminescent and Fluorescent Plate Readers
Richard A. Moravec, Andrew L. Niles, Terry L. Riss, James J. Cali,
Peter F. Stecha, Brian D. Almond and Erika M. Hawkins
We have developed methods to combine fluorescent and luminescent
cell-based assays to measure more than one parameter from a single
sample using ordinary microplate readers. Measuring two or more
parameters from the same sample of cells is more efficient, improves
consistency compared to using parallel samples, and provides
additional information about cellular response. By understanding the
assay chemistries and detection parameters, many different assays
can be combined in a simultaneous, sequential, or split sample
format. We have been successful in multiplexing simultaneous
homogeneous fluorescent and luminescent assays for cell viability,
caspase activity and total cell number in the same well. Additional
homogeneous multiplex examples include real time Renilla luciferase
reporter gene assays followed by measurement of cell viability or
caspase activity. We can overcome assay chemistry incompatibility
for situations such as such as luminescent cytochrome P450 and
luminescent cell viability assays by splitting the sample and
transferring cell culture supernatant to a separate well. While each
assay has individually been proven to be successful in a high
throughput format, we have continued to improve protocols that
enable multiplex detection of more than one indicator from a single
sample. |
