consistency in counting cells by flow

Dr Simon Watson Simon.Watson at ed.ac.uk
Tue Mar 19 06:23:46 EST 2002


Hi Yoav

I'm using Exalpha Perfectcount microspheres for enumeration.
These contain two populations of beads with different sizes and
fluorescences; 50% low and 50 % high fluorescence in all
channels.  The point is that unless the beads are properly mixed,
you won't get a 50:50 mix.  The only problem is if you run a sample
for a long time the brighter (and larger) beads tend to settle.They
need a really good mix immediately before use.  The concentration
is determined by the manufacturers although I tend to confirm this
by manual counting.

You may not need anything as fancy as this and there are a
number of other products available.

Good luck

Simon Watson

On 15 Mar 02, at 14:52, Yoav Altman wrote:

>
> Dear Flowers,
>
> A colleague of mine wishes to use a FACScan to count cells for an
> experiment.  The experiment will proceed over several weeks, and he
> wishes to count live and dead cells over several time points (eg. 1
> wk, 2 wks, etc.)
>
> Assuming that his samples are resuspended in the same volume every
> time, and that the flow rate would be the same from week to week or
> sample to sample, he could just take the number of events in his live
> gate vs. non-debris events.  But, he is concerned that flow rate may
> vary from week to week due to small obstructions in the fluidics.
>
> It seems that if we had a bead of known concentration we could add
> these to the sample and do something like stop collecting after
> 10,000 beads and thus know we had the same volume as last time.  Does
> this seem like a viable approach.  Does anyone make beads in known
> concentration, or is there some other standard we can use to know
> that we are counting cells in the same volume from week to week.
>
> Thanks,
>
> Yoav
> ---
> Yoav Altman
> Imaging Facility Specialist
> La Jolla Institute for Allergy & Immunology
> 10355 Science Center Drive
> San Diego, CA 92121
>
> email: <yaltman at liai.org>
> Phone: (858) 678-4608
> Fax: (858) 558-3526




Dr Simon Watson
MRC Clinical Research Fellow
Phagocyte Lab
First Floor
Medical School
Teviot Place
Edinburgh
EH10 4EB
UK

Tel: +44 (0)131 651 1570



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