Antwort: consistency in counting cells by flow

eug.kreysch@t-online.de eug.kreysch at t-online.de
Tue Mar 19 10:11:32 EST 2002


Hi Yoav,
we have used Becton Dickinsion´s TruCount beads (defined number of beads in
FACS tubes, see http://www.bdbiosciences.com/immunocytometry_systems/) with
good results and calculated absolute cell numbers from total events (e.g.
10,000 to 15,000) using cell and bead counts within the mixture after
gating on beads and PI (propidium iodide) negative live and/or PI positive
dead cells mainly according to BD´s protocol. However, for an accurate
calculation proper mixtures/proportions of beads and cells (not to many
cells!) are necessary.
The major disadvantage is that - as far as I know - BD does not offer the
beads separately under normal conditions. Nevertheless I would ask .....
Best regards
Georg Kreysch





yaltman at liai.org am 15.03.2002 23:52:01

An:   cytometry at flowcyt.cyto.purdue.edu
Kopie:

Thema:    consistency in counting cells by flow


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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