Posted 31 July 2002 - 05:30 AM
Yeah, its quite easy with the ficoll-hypaque method. Dilute your buffy coat 1:2 with PBS. Prepare some 50 ml tubes with 25 ml ficoll (density 1.077 g/ml), then overlay the ficoll with an equal amount of the mixture from above. Centrifuge for 30 min with 700 x g without brake at 4 degress C. Soak up the white interphase between the plasma fraction and the ficoll fraction with a pipette and transfer it into a fresh tube. Wash twice with PBS. Then resuspend the cell pellet in medium and transfer the whole stuff to cell culture dishes (about 5 million PBMC per ml). The resulting PBMC suspension contains about 10% monocytes and 90% lymphocytes. Let the monocytes adhere for 1 h and wash away the non-adherent lymphocytes, so you get a nearly pure monocyte culture.