RE: lysing mouse RBCs

Hi Lori,

Density separation of RBCs is much nicer than lysis when you want to stain your cells for
FACS afterwards. However, Ficoll-Paque is made for human blood and not mouse so the
density is less. Consequently you variably lose white blood cells in a temperature
dependent manner (density changes). So, in order to have consistent white blood cell
preps with good RBC depletion you should use Lympholyte M (from Cedarlane) which is
formulated especially for mouse blood at the correct iso osmolarity (0.168 instead of
0.147). Lower isoosmolarity will decrease the density of mouse blood cells.
If you use this you will have much more consistent cell preps from blod without RBCs.

Anne

Dr Anne Wilson,
Ludwig Institute for Cancer Research,
Lausanne Branch
Epalinges
CH-1066
Switzerland

-----Original Message-----
From: RICE,LORI P [mailto:lrice@ufl.edu] 
Sent: mercredi, 13. février 2008 07:25
To: cyto-inbox
Subject: lysing mouse RBCs


Hi,
I recently posted a similar inquiry and did not get any responses. 
We are also trying to get rid of RBCs in about half ml of whole 
mouse blood. The difficultly is getting rid of the nucleated RBCs, 
which are much more prevalent in murine blood than human and do 
NOT lyse.  We have used PharmLyse instead of FACSLyse to avoid 
using fixatives. We also used the lysis protocol of eBioscience.  
We tried staining before and after lysis.  All of these protocols 
resulted in a lot of residual RBCs and non-specific staining, as 
determined by using TER119 for RBCs and CD45 (we are interested in 
the TER119 neg/CD45 neg population).  Out of frustration, we went 
back to Ficoll-Paque Plus, a product that was recommended to us 
for mouse blood.  This removes more RBCs with less damage, but the 
results staining before or after Ficoll separation varies 
dramatically. Does anyone have any insight into this?

If you are looking for the CD45 positive population, try the good, 
but expensive SpinSep kit from StemCell Technologies.

Lori


--
Lori Rice, Ph.D.
University of Florida
lrice@ufl.edu