neutrophil isolation

[Michael J. Richards]

I have used this gradient some years ago for chemotaxis studies and obtained
very pure neutrophil preps. As I recall you first dilute the anticoagulated
whole blood 50/50 with 150mM saline and overlay your ficoll-hypaque gradient and
after spin collect your bottom pellet which is neutrophil/RBC mix and then lyse
off the RBC's

Jeffrey & Sandy Marion wrote:

> Eric Park wrote:
> >
> > I'm encountering some problems when I try to isolate neutrophils from my
> > blood.  I'm using a Ficoll-Hypaque solution when I centrifuge the blood.
> > This technique works great for several other people but not for me.  A lot
> > of RBCs are remaining in the neutrophil layer and not settling down to the
> > bottom of the tube.  Does anyone know why this is happening to my blood?
> >
> > Personal info:
> > Blood type is B+ Rh+
> > Asian male 22 years old
> >
> > Insights would be helpful since this is a mildly annoying problem in my
> > research by begging for volunteers instead of tapping my own blood.
>
> In the world of flow cytometry, many technologists utilize ficol
> histopaque medium to recover lymphocytes from anticoagulated whole blood
> or lymphocytes from lymph nodes.  Spun at 1500 rpms for 30 minutes
> yields an excellent recovery of these T and B cells with very little
> monocyte or other leukocyte contamination.

--
Michael J. Richards
Research Biochemist