Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.
The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.
Braet
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Faculty of Medicine and Pharmacy, Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Brussels-Jette, Belgium. filipbra@cyto.vub.ac.be
Vermijlen
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Bossuyt
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De Zanger
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Wisse
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D000818:Animals; D003110:Colonic Neoplasms / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D007694:Killer Cells, Natural / Q000276:immunology* / Q000648:ultrastructure; D008099:Liver / Q000276:immunology*; D008297:Male; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar