Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study

Abstract

The colorimetric MTT assay based on the selective ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide into formazan was adapted for measuring antibody independent monocyte mediated cytotoxicity. In view of the suggested application of adoptive immunotherapy we studied the anti leukaemic effects of activated human monocytes and macrophages against U 937 cells in vitro. Purified monocytes (greater than 98%) isolated by centrifugal elutriation and activated with interferon-gamma (IFN) were incubated with U 937 cells for 24 h at effector-to-target cell ratios (E/T) of 0.1-10. We assayed cytotoxicity by relating the optical density (OD) of residual metabolically active U 937 cells after exposure to effector cells to the OD of the initially inoculated U 937 cells. MTT reduction of effector cells was dependent on monocyte activation and differentiation into macrophages but did not interfere with the target cell signal up to an E/T ratio of 10. Improved signals could be obtained by dissolving formazan in DMSO with the addition of glycine instead of using propanol as solvent. Maximum cytostasis (95%, conventional [3H] incorporation assay) and cytotoxicity (80%, modified MTT assay) was reached with IFN activated monocytes at an E/T ratio of 10. In conclusion these data show that the modified MTT assay is a useful method of measuring monocyte mediated cytotoxicity in a sensitive, rapid, semi-automatic manner without the use of radioactive isotopes.