CARE-LASS (calcein-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity.
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.
Lichtenfels
R
R
Department of Neurology, Medical School, University of Tübingen, Germany.
Biddison
W E
WE
Schulz
H
H
Vogt
A B
AB
Martin
R
R
D002857:Chromium / Q000032:analysis; D002860:Chromium Radioisotopes; D003602:Cytotoxicity, Immunologic; D000939:Epitopes; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D008297:Male; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D002860:Chromium Radioisotopes; D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D002857:Chromium; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C007740:fluorexon