RE: lysing mouse RBCs

Hi Lori,

I used to run mouse blood samples all the time and I've found that the best
way (by far) to get rid of red cells is to collect the mouse blood directly
into a 1 mL solution of PBS with 10mM EDTA and 1% dextran (T500).  Mix the
tube well (glass works best) and keep at 37 C for 30-45 minutes.  

The dextran causes the red cells to settle nicely at the bottom of the tube
and the white cells stay above them.  Simply take the supe off the top
(avoiding the pile of red cells), transfer to a flow tube, dilute in
staining media and spin 2000 RPM for 5-10 minutes.  This will get rid of
more than 90% of your red cells.  Then follow with lysing and the remaining
red cells will be history.  These extra steps (although a little more time
consuming) result in beautifully clean data. 

Melissa 

-----------------------------------------------
Melissa R. Makris
Flow Cytometry Lab Supervisor
Virginia Tech
College of Veterinary Medicine
Duckpond Drive, Phase II 0442
Blacksburg, VA 24061
(540) 231-4115
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-----Original Message-----
From: RICE,LORI P [mailto:lrice@ufl.edu] 
Sent: Wednesday, February 13, 2008 1:25 AM
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