CARE-LASS (calcein-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity

Abstract

CARE-LASS is a highly sensitive, fast, simple and safe fluorometric microassay. Target cells are loaded with acetoxymethyl ester of calcein (calcein-AM) that passively crosses the cell membrane. Intracellular esterases convert the molecule to calcein, a polar fluorochrome which, in cells with intact plasma membranes, displays good retention characteristics and low pH sensitivity. In analogy to standard 51Cr release assays, the CARE-LASS system is based on the release of a marker into the supernatant that is measured by an automated fluorescence scanner and correlates with the number of lysed cells. We tested the CARE-LASS system by measuring cytotoxicity in major histocompatibility complex (MHC) class I and MHC class II restricted cytotoxic T lymphocyte (CTL) assays as well as lymphokine activated killer (LAK) mediated cytotoxicity. We applied a small set of target cell lines at various effector to target (E:T) ratios, at different antigen concentrations and compared CARE-LASS CTL data to data resulting from conventional 51Cr release assays. The CARE-LASS system provides a reliable and sensitive method to measure cell-mediated cytotoxicity.