Use of fixed cells in cell contact-dependent cytotoxicity assays for TNF: a cautionary report

The use of fixed effector cells or cell membrane preparations in assays to study cell surface TNF-mediated immunological effects has been widespread for more than a decade. The assumption has always been made that observed effects of fixed cells are due to the cell surface TNF molecule. Here we report that paraformaldehyde-fixed, LPS-stimulated RAW264.7 cells release a factor that causes cytotoxicity against TNF sensitive WEHI-164 cells. The factor was neutralized by soluble TNF receptors as well as antibody and could not be removed by ultracentrifugation at 100,000 x g, and is therefore likely to be a form of soluble TNF. This has important implications for the interpretation of these assays, given that cell surface TNF is thought to exert biological effects through a different signaling mechanism than soluble TNF.