FW: Cell Volume Revisited

MATTHEW ROSINSKI mrosinski at cheque.uq.edu.au
Fri Oct 13 22:26:59 EST 2000


I agree that for cell sizing the Coulter really is the
only option.  Nucleated cell probably give more
interference in laser light scattering techniques than
possible with a Coulter.  To use a flow cytometer to get an idea of
cell volume it would probably be better to use a protein stain
eg: FITC rather than FSC.  FSC does not correlate very well
with Coulter volume especially when the cells are under-
going changes (resulting in differing scattering properties).
Protein content however does generally correlate directly
with cell size during balanced growth.
(Frame and Hu, 1990, Biotechnol. Bioeng.36: 191-197)

Measuring the Coulter volume one should always be sure
to be careful of the Osmometer effect.  Especially when
the sample is derived from a system of unknown or undefined
osmolality.  eg:diluting cells in a solution of higher osmolality than
the source will generally lead to immediate shrinkage and
measurement of a volume which doesn't truly represent that
of the source.  (Rosinski, 2000, Biotechnol. Prog. 16: 782-785)

Incidentally, regarding Jim's message I think smaller plastic beads
actually scatter light more widely than larger ones
according to Mie Theory.  What is measured in the FSC
PMT is probably a stronger signal for larger particles
because they FAIL to scatter the incident light as much
as smaller particles.  Perhaps someone can clarify?

Matt Rosinski
_______________________________________________________

Matthew Rosinski
The University of Queensland
Department of Chemical Engineering
College Rd
St Lucia  Q  4072
Australia
Ph: (07) 3365 8392/4352

_____________________________________________________

-----Original Message-----
From:	James Weaver 301-594-5879 FAX 301-594-3037 [SMTP:WEAVER at CDER.FDA.GOV]
Sent:	Friday, October 13, 2000 3:55 AM
To:	Cytometry Mailing List
Subject:	Re: Cell Volume Revisited


Despite what is written in various places, flow cytometry can only be
used for size measurement on homogenous particles such as plastic beads.
It does not work for particles with complex optics such as cells. With
beads, increasing size leads to increased light scatter. With cells the
situation is quite different, a treatment that resulted in a 30% change
in cell volume caused a light scatter decrease in a lymphoid cell line
and no change in light scatter from NIH3T3 cells.

	Coulter volume measurements are regarded as the gold standard of
cell volume measurements. I have done cell volume measurements on
nucleated cells in the past with no problems. If you are not seeing
changes in cell volume, than either the treatment is not causing a
change or the instrument needs repair.

-Jim Weaver


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