75Se-release: a short and long term assay system for cellular cytoxicity.

Abstract

The gamma-emitting aminoacid 75Se-selenomethionine (75SeM) was examined as a target cell label in cytotoxic assays. It was efficiently taken up by activated, intensively metabolizing cells of various types but hardly at all by resting or low-metabolizing cells. Culturing activated cells in methionine-deficient medium with 3--5 mu Ci75SeM/ml for 18--22 h usually resulted in an uptake of 3--20 cpm/cell which was 3--200 times that of 51Cr marked cells. 75SeM-labelled cells kept in medium at ambient temperature or at 37 degrees C, maintained a high radioactivity per cell and a viability above 85% for at least 72 h without significant increase in spontaneous isotope release or loss in sensitivity in subsequent cytotoxic tests. 75Se-labelled material released from target cells was not reutilized by unlabelled lymphoid cells. Provided the cells were carefully washed after labelling and kept in optimal culture conditions, the reasonably low baseline release (usually 0.6--1.8% of input/h) in the medium control allowed performance of long-term assays of up to 54 h. However, strong cytotoxic reactions (e.g. ADCC) could cause over 50% specific 75Se-release within 5 h. With constant amounts of effector cells (3.6 x 10(3) up to 3 x 10(5)/well) identical or even higher, specific releases were obtained on 6 x 10(2) targets as compared to 1 x 10(4) targets/well. Thus, the 75Se-release assay offers a single monitoring system suitable for short (3--6 h) and long term (usually up to 44 h) cytotoxic reactions on a microscale, using 1 x 10(3) or less targets/well. Its sensitivity permits evaluation of strong and weak reactions as well as early and delayed onset cytotoxicity. In addition, with a gamma-spectrometer the radioactivity of 75Se can easily be distinguished from that of 51Cr. Due to this, and an improved method for 51Cr labelling of cells (10 mu Ci 51Cr/ml medium for 18--22 h), a double gamma-labelling of cellular proteins is available which provides new possibilities for monitoring cellular interactions in short and long term tests.