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 f or more than a decade. The assumption has always been made that observed ef fects of fixed cells are due to the cell surface TNF molecule. Here we repo rt that paraformaldehyde-fixed, LPS-stimulated RAW264.7 cells release a fac tor that causes cytotoxicity against TNF sensitive WEHI-164 cells. The fact or was neutralized by soluble TNF receptors as well as antibody and could n ot be removed by ultracentrifugation at 100,000 x g, and is therefore likel y to be a form of soluble TNF. This has important implications for the inte rpretation of these assays, given that cell surface TNF is thought to exert biological effects through a different signaling mechanism than soluble TN F. (C) 1999 Elsevier Science B.V. All rights reserved.