Is the increased absorbance over the whole plate of samples or just in random individual wells or individual sets of
replicates? And is it a slight increase in absorbance or are the 'false' positive wells much darker than the zero TNF
(untreated) control? Just ruling out bacterial contamination of your samples which can occur in random wells (probably assay
technique) or in sets of replicates (probably sample preparation and storage) and which can give off the scale high absorbance.
Although I guess that if it's the same individuals who have carried out the assay previously it is unlikely to be either of
the above solutions. It's worth checking the wells under a microscope just in case tho'.
If the increase absorbance is not great in the 'false' positives is it possible that there is some cell proliferation or
simply less TNF in the rat plasma? I haven't done it myself but you could try spiking the samples that give consistant
'false' positives with TNF at a concentration that gives about 50% killing and see if the cells show the same absorbance as the
same TNF concentration on your standard curve. If it's low TNF conc. in your sample then spiking it you would expect a
result similar to the same TNF standard concentration, and if its something in the sample protecting the cells or causing
proliferation then the response to that concentration of TNF would differ from that of the standard. A dose response curve
would be the ideal but I assume your sample volumes are not great so spiking one set of replicates might give you some idea of
the problem's cause.
Finally I would say that some of the original clones of WEHI and I _believe_ L929 cells tended to lose their sensitivity to TNF
over time but I suppose that would have affected your standard curve as well.
Good luck,
Ben Zeitlin
Division of Biomedical Science
Sheffield Hallam University
UK
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