Thursday, August 2, 2012

Applications Thursday: Alternative Cell Toxicity TRF assay for the Radioactive Chromium (CrVI) Release Assay

Principle of the DELFIA® cytotoxicity assay

There are many ways to study cell toxicity and apoptosis when using homogenous cell populations, but there are limited ways when using mixed cell populations. One common example of cell death in a mixed cell population occurs in the process termed immunosurveillance in which cells of the immune system, such as cytotoxic T lymphocytes (CTLs) and Natural Killer (NK) cells, induce apoptosis of target cells. Assaying apoptosis in mixed cell populations usually requires the cells of interest to be pre-loaded with a reagent, and the loss of membrane integrity that occurs during cell death results in the reagent being released. The most widely used assay is to pre-load cells with radio-labeled chromium. However, 51Cr is an unstable photon emitter and requires costly and cumbersome lead shielding for safe use.

As alternative to the radioactive chromium release assay, a microplate reader is used with a time-resolved fluorescence assay to study the loss of membrane integrity. In this assay, target cells are loaded with fluorescence enhancing ligand, BATDA, which penetrates the cell membrane quickly and is hydrolyzed to form a hydrophilic ligand (TDA) that is released from cells only after cytolysis (Figure). In the presence of a solution containing Eu3+, the released TDA forms a highly fluorescent and stable chelate (EuTDA), whose levels are measured using the time-resolved fluorescence mode of the PHERAstar microplate reader. It is shown that this assay produces results that are similar to the classical chromium release assay.

See more about performing this application on the BMG LABTECH PHERAstar microplate reader here: http://www.bmglabtech.com/application-notes/time-resolved-fluorescence/defia-cytotoxicity-assay-192.cfm. This application can also be performed on the FLUOstar or POLARstar Omega microplate readers.

Delfia is a registered trademark of Perkin Elmer Inc.

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