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Determination of Cytotoxic Activity of TNF-α Using Colourimetric Microassay and Electron Microscopy.

1Gamal E. Eid and 2Sameh E. El-Shewemi.

1Microbiology & Immunology Unit and 2Histology & Electron Microscopy Unit Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh.

Tumor necrosis factor-alpha (TNF-a) is one of the major cytokines regulating a large spectrum of physiological and pathophysiological functions of the immune system. In this investigation, a colourimetric microassays and electron microscopy were used to study the cytotoxic effect of TNF-a on WEHI-13 VAR (mouse fibrosarcoma) as tumor target cells. Water-soluble tetrazolium (MTS/PMS) reduction as well as crystal violet (CV) staining assays were used to detect the cytotoxic activity induced by different TNF-a concentrations (1, 10, 100, 1000, and 10,000 pg/ml) and incubation times (0, 1, 2, 4, 6, 9, and 20 hours). Cytotoxicity started to develop after 9 hrs of incubation and reached 100 % at 10,000 pg/ml after 20 hrs of incubation when detected by MTS/PMS assay and 86.1 % when detected by CV assay. Although, the MTS/PMS method expressed higher sensitivity than CV staining assay, the percentage of cytotoxicity induced by TNF-a was directly correlated to each other when detected by both colourimetric assays. The ultrastructural changes induced by TNF-a on target cells were mainly cytoplasmic rather than nuclear. These changes were also dependent on the dose and / or duration of exposure to TNF-a.