PBMC Isolation

I am isolating human PBMC using Ficoll. > nearly 70% of separations result in PBMC clumping together and either

RBC or forming a 'membrane' at the interface of the ficoll/buffer.  does anyone have a recipe? 

Are you getting a 'buffy coat' - a brown-grey mass of cells at the interface? Ensure that you are using hypaque 1077 or other Ficoll gradient at ~24dC, with very careful Ficoll underlay/medium overlay, and centrifugation at ~1350g for ~7 mins, no brakes. Following this round of centrifugation, get the interface layer which is enriched for PBMCs, and wash with 8-10x vol HBSS/culture medium  (i.e. for 1.5 ml of interface removed, fill the 15 ml tube with HBSS) at 350g for 5 mins. When this cell pellet is resuspended, you'll end up with a nice single cell suspension.
As for the RBCs, thats probably a direct consequence of overloading the gradient. If you still see a very red pellet at the wash step above, just perform a Gey's or AmmCitrate lysis for RBCs.

Also, check out the "related topics' feature on the right panel alongside this post for previous queries, and perform a site search for additional issues, and detailed protocols.

Hello
Thanks for your considerations. I will follow your protocol saturday. I hope this works. ithink my problem is due to Ficoll-paque. I will try your suggestions. I will inform you.