Mix fresh EDTA blood with an equal volume of room-temperature PBS in a conical centrifuge tube. 2. Slowly layer 3 ml of Ficoll-Hypaque under every 10 ml of the blood/PBS mixture. 3. Centrifuge the mixture at 900 x g for 30 minutes at room temperature with no brake. 4. Carefully remove the upper plasma layer and transfer the mononuclear cell layer to a new tube. 5. Wash the cells three times with PBS (3x volume of the cell layer), centrifuging each time for 10 minutes at 400 x g. 6. Resuspend the cells in complete RPMI-10 for immediate use or 10% DMSO for freezing.