Ficoll-Hypaque density can be adjusted (increased) to allow separation
of pmn *and* mnc from rbc of whole blood. There are several commercial
preparations.
The dilution and size of tubes also effects separation.
I worked with someone who used COLD F/H to isolate his pmn, but he must
have modified the protocol (temp effects fmlp response). He then used
water to lyse residual rbc.
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.
>
Dom Spinella wrote:
It's not uncommon for the Ficoll-Hypaque interface to have some red
cells in it. You an reduce (but not eliminate) them by diluting the
blood sample in isotonic buffer prior to layering on Ficoll-Hypaque. If
the remaining rbcs are a problem, you can lyse them by brief incubation
in Tris-buffered ammonium chloride (e-mail me if you need a recipe).
BTW, the Ficoll-Hypaque interface contains PBMCs -- peripheral blood
mononculear cells which include other cell types besides just
neutrophils (monocytes and lymphocytes for example). Cheers. --Dom
Spinella
---> Dennis
**********************************************************************
Dennis J. Young Voice : (619) 822-0407
Flow Cytometry Core Facility FAX : (619) 822-0412
University of California, San Diego <mailto:djyoung at ucsd.edu>
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