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Abstract for Homogeneous Fluorescent In Vitro Toxicity Assay
that Measures Cell Membrane Integrity
Terry Riss, Rich Moravec, Nancy Murphy and Deborah Bishop
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
We have developed a homogeneous fluorescent cytotoxicity assay
that measures the release of lactate dehydrogenase (LDH) from cells
with damaged membranes. The novel “add and read” homogeneous format
of the CytoTox-ONE™ Assay (patent pending) was enabled by
developing assay conditions that do not damage living cells, thus
only LDH present in culture medium is detected and viable cells do
not contribute to the signal. The CytoTox-ONE™ Assay procedure is
to add reagent directly to cells, incubate for 10 min, stop the
reaction, and record fluorescence. There is a linear relationship
between the fluorescent signal and the number of damaged cells. The
assay can detect fewer than 200 damaged cells in a 384-well format.
The release of LDH from cells correlates inversely with viable cell
number. The stability of the reagents and fluorescent signal are
compatible with automation of the assay for HTS. Multiplexing of the
CytoTox-ONE™ Assay with other assays will be described. |
