Ficoll-Hypaque density can be adjusted (increased) to allow separation
of pmn and mnc from rbc. The dilution and size of tubes effect
separation.
I worked with someone who used COLD FH to isolate his pmn, but he must
have modified the protocol. He 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?
>mark wrote:
> First the others had good answers vis a vis Temp and dilution etc but i was
> under the impression that f/h centrifugation results in mononuclear cells
> and most granular/polymorphonuclear cells are not inthe interface which is
> what i thought a neut was, its a monocyte?
>> there is definately a problem if blood is cooled don't do that. also some
> people's blood does act slighly differently in the technique. for example,
> sickle cell or thalassemia RBC's don't work very well in f/h centrifugation.
> But be the vampire and use all other possible people-- give the grads student
> a $20 (covered by you r dept of course) and you probably won't have a problem
> gettin blood
> good luck
>
---> 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>
**********************************************************************
C. J. Fuller wrote:
>> In article
> <Pine.A41.3.96.990204142406.62208A-100000 at node5.unix.Virginia.EDU>, Eric
> Park <yhp4n at node5.unix.Virginia.EDU> 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.
>> Eric-Are you warming up the Ficoll-Hypaque solution before you layer on
> the blood? The solution should be at room temp, and your blood should be
> cooled down, before you layer on the blood.
>> In our lab we separate the neutrophils from the RBC's with Dextran
> sedimentation after getting rid of the other WBC's, then use hypotonic
> lysis to get rid of any stubborn RBC's.
>> You may want to try this, but you may want to beg a little blood from
> others, too. You can't write a thesis with an N of 1.
>> Good luck!
> Cindy Fuller, PhD, RD
>> --
> C.J. Fuller
> <mailto:cjfuller at erickson.uncg.edu>
> <mailto:cjfuller at mindspring.com>
--
---> 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>
**********************************************************************
