Measurement of cytotoxicity by propidium iodide staining of target cell DNA. Application to the quantification of murine TNF-alpha.
A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-alpha), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane permeable due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5X10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-released assays that kinetic studies of TNF-alpha toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-alpha.
Wrobel
K
K
Universidad de Oviedo, Departamento de Biología Funcional, Facultad de Medicina, Oviedo, Spain.
Claudio
E
E
Segade
F
F
Ramos
S
S
Lazo
P S
PS
D016475:3T3 Cells; D000818:Animals; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects*; D004247:DNA / Q000032:analysis*; D005453:Fluorescence; D051379:Mice; D011419:Propidium; D013194:Staining and Labeling; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D004247:DNA