Re: stains AFTER RBC lysis.

Hello Uriel,

I was wondering if you would consider using Dextran T-500 (6% solution 
in PBS/10mM EDTA) to first sediment the RBCs from your whole blood.  I 
have found this to be effective when I was working with eosinophils and 
basophils in humans and a number of other species.  The whole blood is 
mixed 2:1 with the Dextran and spun at the lowest possible force you can 
run on your centrifuge.  RBCs should sediment at room temperature while 
leucocytes stay in the buffy layer. 

Should you wish to get rid of remaining RBCs, I had further luck with 
using 0.2% NaCL, ice cold, for 45 seconds.  Equilabrate this with ice 
cold 1.6% NaCL.  It is a bit gentler than water and Ammonium chloride 
solutions.

I hope that this helps. 

Eric


Uriel TK wrote:

> Dear friends:
> The subject of RBC lysis has been touched extensively but i haven't 
> found the reason why in the protocols invariably staining is done 
> before RBC lysis and not after. We need to acquire a lot of leukocytes 
> to reach a good number of blood DCs, some 1/2 to 1 million, so i need 
> to concentrate the cells before staining (100 micoL of blood do not 
> have enough leukocytes). We will be working with patient blood, some 
> mLs only so i cannot use ficoll (also ficoll has been reported to 
> affect the relative numbers of leukocyte populations or expression of 
> markers). We will use water or ammonium based lysis. Is there a 
> pitfall or specific problem i should be aware of? How do you deal with 
> RBCs when looking for rare events in blood?
>
> Thanks in advance,
> Uriel.
>
>