Re: Absolute counts from flow cytometry

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
15 Nov 1996 15:04:41 Z

Mouse blood can indeed be harder to lyse but most hematology
analyzers have been adapted for different species using
different settings. For small frequency throughput you are
better off using a non-wash lyse and spike your sample with
beads of known concentration if you can't do a volume count.
If you use a fixative lyse you can add PI, if you use
distilled water you can use lds751 , thiazole orange, or any
of the supravital stains on offer for better discrimination.
Even better you measure without lyse. Perhaps Molecular
Probes 'buck-count' works for that purpose already,
otherwise I am sure they will make up a cyto-count kit for a
few more bucks.

Gerhard.nebe-von-caron@unilever.com

______________________________ Reply Separator _________________________________
Subject: Absolute counts from flow cytometry
Author: Jeff_Carrell@hgsi.com at INTERNET
Date: 15/11/96 04:01

We are trying to generate absolute counts for different cell types in the
peripheral blood of mice. What are your preferred methods?

I understand that one common practice is to multiply the WBC (from an automated
cell counter such as a Coulter Counter) by the percent staining (from FACS).
However, we are questioning the validity of this approach because of the
different lysing methods used for the two types of instruments. Some non-
erythrocytes may be lysed in different proportions with each method. Is
multiplying this WBC count by a relative percentage accurate?

Of course, these instruments were designed for human blood, so trying to adapt
procedures for mice is a challenge...but it shouldn't be impossible. Any
thoughts would be greatly appreciated.

Jeff Carrell
Human Genome Sciences