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.
Watts
A D
AD
Department of Pathology, University of Sydney, NSW, Australia.
Onier-Cherix
N
N
Hunt
N H
NH
Chaudhri
G
G
D000818:Animals; D002450:Cell Communication / Q000276:immunology; D003168:Complement Fixation Tests; D003602:Cytotoxicity, Immunologic; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D011108:Polymers / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D005404:Fixatives; D008565:Membrane Proteins; D011108:Polymers; D014409:Tumor Necrosis Factor-alpha; D005557:Formaldehyde; C003043:paraform