Monocyte Isolation from Whole Blood via Gradient Centrifugation - (Oct/04/2010 )

I am trying to isolate monocytes from whole blood at a very high purity to eventually be transfected with siRNA. I have tried several different methods including :cold aggregation, adherence, and gradient centrifugation with diluted Percoll. All have resulted in low yield with significant lymphocyte contamination. My goal is to achieve 90% purity and then use a CD14 magnetic bead negative isolation. Ideally, I would like a yield to be between 50-70 x 10^6 cells.

I use Histopaque 1077 from sigma to obtain mononuclear cells from a buffy and then I do positive selection with CD14 (miltenyi) I tried percoll before and obtain few cells and the same with adherence.
Good luck