In article <Pine.SOL.4.05.9902121021020.5801-100000 at granby>, Carol Iversen
<rczci at unix.ccc.nottingham.ac.uk> wrote:
> I am isolating human PBMC using 'Lymphoprep'. Some times it works but
> nearly 50% of separations result in PBMC clumping together and either
> sinking to sit on the RBC or forming a 'membrane' at the interface of the
> ficoll/buffer. I have tried using PBS and RPMI, I have heard a citrate
> buffer may prevent this - does anyone have a recipe? Or any other advice
> or explaination why this happens?
I use Ficoll-Hypaque myself, and have few problems unless the blood was
rich in fat (when blood was drawn shortly after the donor ate). Also, the
blood should be diluted at least 1:3 with some los-viscosity buffer (cell
culture medium of PBS) prior to layering it on top of the Ficoll.
Some samples show minor clumping / 'membrane formation' when put into
another medium, eg when done washing the cells after isolation. This
cannot be prevented as far as I know simlply because the reason for this
is unknown (although I feel it might be DNA from accidentally lysed
cells). Don't bother pipetting the clump, because it won't release at all.
Still, it only affects a very small part of the sample in our hands.
Guy
--------Guy Hermans, PhD student------------
MS research Unit Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute Fax 0032 (0) 11/26.92.09
Belgium
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