consistency in counting cells by flow

Yoav Altman yaltman at liai.org
Thu Mar 21 18:48:12 EST 2002


Flowers,

Thanks to all for the many helpful suggestions.

Here's a brief summary of the responses:

Most agreed that using beads would be the best way to do this-
probably what we'll do.  Beads mentioned included: BD, TruCount;
Beckman Coulter, Flow Count; Spherotech, AccuCount;  Immunotech, Flow
Count; Exalpha, Perfectcount.

Some suggested that knowing the bead concentration was irrelevant so
long as the same volume from the same suspension is used with each
sample.  Beads will stick to pipet tips and tubes, so consistent
techniques must be employed.

A couple of people pointed out that counting dead cells is
problematical because you don't know how many of them end up as
debris.

A couple of respondents suggested using a hemocytometer, and one
individual suggested using CFSE labeled cells from a cell line of
known concentration.

Thanks again for the speedy help,

Yoav



At 2:52 PM -0800 3/15/02, 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



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