Improvement of a cytokine (TNF-α) bioassay by serum-free target cell (WEHI 164) cultivation.

Cytotechnology
Y FigenschauK Bertheussen

Abstract

The elaboration of a sensitive bioassay for assessment of tumour necrosis factor alpha (TNF-α) in a defined medium is described. The assay is based on the cytotoxic effect of TNF-α on a target cell line, the murine fibrosarcoma WEHI 164 clone 13. Cytotoxicity was assessed by detecting the rate of tetrazolium salt reduction employing a spectrophotometer (ELISA-reader). A similar bioassay was used previously to assess TNF-α, though this was dependent on cell growth in a medium containing serum. By employing a synthetic serum replacement, the WEHI cells were adapted to growth in a defined medium which allowed both the propagation of the cell line and the assay to be performed under completely defined conditions. Thus, factors in serum that may influence the TNF-α assessment, such as growth factors, cytokines, soluble cytokine-receptors and macroglobulin, were avoided. The only protein required in this bioassay was insulin, while albumin was added as a carrier protein and to protect the cytokine against loss of biological activity during multiple freeze and thaw cycles. The present assay was optimised to achieve a high sensitivity and, by testing endogenous TNF-α originating from the macrophage-like cell line RAW in both the serum-...Continue Reading

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Citations

Oct 19, 2011·Clinical & Developmental Immunology·Kadri Haller-KikkataloRaivo Uibo
Dec 14, 2017·Journal of Veterinary Emergency and Critical Care·Kris GommerenDominique Peeters
Dec 24, 2021·Microbiology Spectrum·Ludmil Benov

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