neutrophil isolation

Dom Spinella dspinella at
Thu Feb 4 15:13:50 EST 1999

> 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.

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

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