Re: [ RBC lysis in frozen samples ]

Dear Janvier,

We use BD FACS Lysing solution, which is supplied as a
10X concentrate. We dilute this to 1 X solution (10ml
plus 90mls of distilled water) and for fresh blood
samples we use 1ml of the diluted for 25ul of blood
samples we stain. 

For frozen samples we also encountered problems with
lysing and some reasonable lysis is achieved by
bringing the frozen samples to room temperature before
staining, and doubling the amount of the 1 X FACS
lysing solution to 2mL, vortexing the mixture
vigorously and incubating at room temperature but in
the dark for 15 minutes. Follow this with two washes
with 1 ml PBS by centrifuging at 1200 rpm. The lysing
is not as good as in fresh samples but it is much
better than when we use only 1 ml.

Good luck

Wilson Mandala, Research Scientist, Wellcome Trust,
Blantyre, Malawi
--- fjmoreno@isciii.es wrote:

> Dear friends,
> 
> 
> We are doing cell immunostaining in whole blood
> samples, when we use
> fresh samples the lysis of the red cells works OK
> but when we use
> frozen samples there are a lot of  red cells that
> are not eliminated.
> What can I do??
> 
> Thanks a lot
> 
> Javier
> 
> 
> 



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