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Abstract for MultiTox-Fluor Multiplex Cytotoxicity Assay
The MultiTox-Fluor Multiplex Cytotoxicity Assay is a
single-reagent-addition fluorescent assay that simultaneously
measures the relative number of live and dead cells in cell
populations. The MultiTox-Fluor Multiplex Cytotoxicity Assay gives
ratiometric, inversely correlated measures of cell viability and
cytotoxicity. The ratio of viable cells to dead cells is independent
of cell number and, therefore, can be used to normalize data.
The MultiTox-Fluor Assay simultaneously measures two
protease activities; one is a marker of cell viability, and the
other is a marker of cytotoxicity. The live-cell protease activity
is restricted to intact viable cells and is measured using a
fluorogenic, cell-permeant, peptide substrate (glycyl-phenylalanyl-amino-fluorocoumarin;
GF-AFC). The substrate enters intact cells where it is cleaved by
the live-cell protease activity to generate a fluorescent signal
proportional to the number of living cells. This live-cell protease
becomes inactive upon loss of cell membrane integrity and leakage
into the surrounding culture medium. A second, fluorogenic cell-impermeant
peptide substrate (bis-alanyl-alanyl-phenylalanyl-rhodamine 110;
bis-AAF-R110) is used to measure dead-cell protease activity, which
is released from cells that have lost membrane integrity. Because
bis-AAF-R110 is not cell-permeant, essentially no signal from this
substrate is generated by intact, viable cells. The live- and
dead-cell proteases produce different products, AFC and R110, which
have different excitation and emission spectra, allowing them to be
detected simultaneously.
The MultiTox-Fluor Assay is designed to accommodate
downstream multiplexing with many Promega luminescent assays or
spectrally distinct fluorescent assay methods, such as those
measuring caspase activation, reporter expression or orthogonal
measures of viability. |
