Mr S.A. Ali wrote:
>> Graham Briars (g.briars at mailbox.uq.edu.au) wrote:
> : While we are on the subject. I have been using the Ficoll Hypaque
> : modification of Ferrante and Thongs method. I regularly get
> : half of the red cells appearing at the bottom of the tube and half
> : suspended about 1mm below the neutrophil layer. It makes pipetting the
> : neutrophils a bit more difficult, but it works. Has anyone else had and
> : resolved this problem.
>> You probably do this anyway, but after pipetting the neutrophil layer,
> we subject the RBC to hypotonic lysis (added water for 10s and then
> sufficient NaCl to make the medium isotonic) and then span the mixture.
> After, we washed the cells a couple of times in something like RPMI or PBS.
In my experience with the Hypaque method, this problem varied with
individuals- some donors always gave a clean separation of RBCs from the
rest, and some always had a big band of red gunk at the interface. You
can lyse the RBCs with hypotonic shock or ammmonium chloride buffer but
it does mean some extra washing.
Ferrante & Thong found that separation depended on the height of the
blood column not its total volume, so use the narrowest tubes you can. I
used to divide 10mls of blood into 4 x 2.5ml lots and spin each over
about 3 mls of Hypaque. This does seem to help. Diluting the blood
doesn't make any difference.
Actually, I have become increasingly disenchanted with these separation
techniques- let's pour all our Ficoll down the sink and do the
experiments with whole blood diluted in medium as nature intended.
Lymphocyte proliferation assays work fine like that, with really low
backgrounds. As for neutrophils, they are 90% of the white cells anyway,
so you can do a phagocytosis assay and be pretty sure that its
polymorphs doing the dirty work.
Andy Fell
