Cell-mediated cytotoxicity for melanoma tumor cells: detection by a (3H) proline release assay.
An in vitro lymphocyte-mediated cytotoxicity assay using [3H]proline-labeled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [3-H]proline by filtering the total culture fluid containing both 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 thus 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 less than 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [3H]proline test was compared with three different 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.
D002460:Cell Line; D002850:Chromatography, Gel; D003601:Cytotoxicity Tests, Immunologic; D006801:Humans; D007111:Immunity, Cellular; D008214:Lymphocytes / Q000276:immunology*; D008545:Melanoma / Q000175:diagnosis / Q000276:immunology*; D011392:Proline / Q000378:metabolism; D012878:Skin Neoplasms / Q000276:immunology*; D013997:Time Factors; D014343:Trypan Blue
D011392:Proline; D014343:Trypan Blue