PBMC isolation by ficoll centrifugation - (Mar/08/2011 )

Hi everybody,
this is my problem: Over years I use ficoll-paque gradient centrifugation to get PBMC from whole blood or buffy coats. 2 weeks ago I started experiencing some difficulties - when overlaying diluted sample over ficoll, it starts immediately (not much, but still) falling to the bottom. The following centrifugation does not give the expected Erc-ficoll-PBMC-plasma layers, but Erc at the bottom and than smooth bloodish-to-finish gradient towords the rim. No strict layers. Originally I blamed very low freezing tempreratures that I thought might have somehow lyze specimen during its transport from the hospital to our lab, but using polystirene box to keep specimen at "room" temp did not help. Since this experience I was only twice able to make good separation out of 8 attempts. :-( Chemicals OK, hands always the same, centrifugation force same, blood samples always different as usually...
I am really clueless since I considered this technique a very simple... and that is may the problem - I do not know what to improve.

I really apreciate ANY suggestions and thank you for reading this.
Paja

are you sure your ficoll is the correct concentration? not decomposed?

have you tried a different lot of ficoll?

have you prepared fresh ficoll?

mdfenko on Tue Mar 8 17:03:39 2011 said:

Hi mdfenko,
Thanks for reply. I tried two different bottles (same lot) but also ficoll by different producer... No change :-( I concluded ficoll is not the problem. But to blame blood and buffy coat specimens is impossible.. :-(