75Se-Release: A Short and Long Term Assay System for Cellular Cytotoxicity

Abstract

The γ-emitting aminoacid ⁷⁵Se-selenomethionine (⁷⁵SeM) was examined as a target cell label in cytotoxic assays. It was efficiently taken up by activated, intensely 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 μCi ⁷⁵SeM/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°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 × 10³ up to 3 × 10⁵/well) identical or even higher, specific releases were obtained on 6 × 10² targets as compared to 1 × 10⁴ 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 × 10³ 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 γ-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 μCi ⁵¹Cr/ml medium for 18–22 h), a double γ-labelling of cellular proteins is available which provides new possibilities for monitoring cellular interactions in short and long term tests.