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Non-replicating toxicity assay

Graeme Price g.e.price at bham.ac.uk
Wed Jun 19 12:43:44 EST 1996

In article <1996Jun19.052839.22761 at marlin.jcu.edu.au>,
nicholas.moody at jcu.edu.au (Mr Nicholas Moody) wrote:

> Hi, 
> I am looking at the effect of an ultraviolet iradiated iridovirus on
> cell culture. The UV irradiated virus does not produce replicating CPE
> after inoculation, however, when the cells are inoculated at a
> relatively high MOI (equivalent to 1 - 5 infectious particles per
> cell) the cells lift of the culture flask.This effect is totally
> different to replicating CPE and is dose-dependent. Staining with 0.4%
> trypan blue (final concentration) to detect dead cells was not
> successful.
> Does anyone know of an alternative to trypan blue staining or any
> other way I might be able to quantify this effect.

I'm not sure about iridoviruses, but this sounds almost exactly like what
we are seeing with influenza virus in MDCK cells. These cells undergo
apoptosis (programmed cell death) when infected with 'flu (about 12-24 h
p.i.). It sounds as though this could be what you are seeing, as cell
rounding and detchment from the monolayer without cell membrane disruption
is seen in apoptosis. To check on this you could use a DNA binding dye
(acridine orange and propidium iodide are both good) on fixed cells and
look for changes in nuclear morphology. If you want to e-mail me for some
methods and bits then go right ahead.

Good luck.


Graeme Price
Microbial Molecular Genetics and Cell Biology Group, 
School of Biological Sciences, Biology West Building,
University of Birmingham,
Edgbaston, Birmingham,
West Midlands, B15 2TT.
United Kingdom.

Tel. (+44) (0)121 414 6555
Fax. (+44) (0)121 414 6557
E-mail g.e.price at bham.ac.uk

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