title: Discordant and anomalous results among cytotoxicity assays: the confounding properties of eosinophil granule major basic protein on cell viability assays.
abstract: When five cytotoxicity methods compared the toxicity of eosinophil granule major basic protein (MBP) and melittin to K562 and HL-60 cells, strikingly discrepant results were noted. Trypan blue staining, propidium iodide/CellTrackerGreen staining and incorporation of 14C-leucine assays indicated MBP damages > 75% of cells by 1 h. In contrast, 51Cr and lactic dehydrogenase (LDH) release assays indicated MBP damages most cells only at 20 h. All methods indicated melittin damages nearly all cells by 1 h. Further studies showed that without cell transfer, dye staining methods indicated MBP produces < 10% cytotoxicity after 4 h. A modified 14C-leucine assay, employing sodium dodecyl sulfate solubilization and trichloroacetic acid precipitation, showed lower cytotoxicity, 48%, at 4 h. Modified 51Cr and LDH assays showed increased cytotoxicities at 4 h, 34% and 58%, respectively. Overall, MBP's ability to cause molecular and cellular adhesion systematically confounds standard cytotoxicity measurements. However, the modified 14C-leucine assay provides a valid measure of MBP's cytotoxicity and may be useful for analyses of 'sticky' cytotoxins.
authors.affiliation: Department of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
authors.forename: H
authors.initials: H
authors.lastname: Kubo
authors.forename: D A
authors.initials: DA
authors.lastname: Loegering
authors.forename: Y
authors.initials: Y
authors.lastname: Tohda
authors.forename: J
authors.initials: J
authors.lastname: Bankers-Fulbright
authors.forename: C R
authors.initials: CR
authors.lastname: Weiler
authors.forename: H
authors.initials: H
authors.lastname: Nakajima
authors.forename: L L
authors.initials: LL
authors.lastname: Thomas
authors.forename: C R
authors.initials: CR
authors.lastname: Adolphson
authors.forename: G J
authors.initials: GJ
authors.lastname: Gleich
mesh_terms: D001798:Blood Proteins / Q000494:pharmacology*; D002448:Cell Adhesion; D002470:Cell Survival / Q000187:drug effects; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology*; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008555:Melitten / Q000494:pharmacology; D012260:Ribonucleases
chemical_list: D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D008555:Melitten; D007770:L-Lactate Dehydrogenase; D012260:Ribonucleases; D007930:Leucine