Cell‐Mediated Cytotoxicity for Melanoma Tumor Cells: Detection by a (3H) Proline Release Assay

Abstract

An in vitro lymphocyte-mediated cytotoxicity assay using [3H]proline-labeled human Mel-ku-77 and Mel-Ei-78 cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [3H]proline by filtering the total culture fluid containing trypsinized tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (P < 0.001) than normal donors'' lymphocytes against 3 melanoma cell lines. In a series of parallel experiments in 36 patients and 18 normal donors, this modification of the [3H]proline test was compared with 3 assays the conventional microcytotoxicity test of Takasugi and Klein, the original [3H]proline microcytotoxicity test of Bean et al. and the viability count of tumor cells.