Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

After collecting 40 milliliters of fresh human whole blood in four 10-milliliter EDTA vacuum tubes, use sterile technique to transfer all of the blood into a single 50-milliliter conical propylene tube in a biosafety cabinet. Following the manufacturer's instructions from a selected human monocyte isolation kit, add 2 milliliters of monocyte isolation cocktail from the kit to the tube of blood, and vortex the magnetic beads from the kit for 30 seconds.

Add 2 millimeters of beads to the blood, and use a 25-milliliter serological pipette to carefully mix the beads with the blood. After five minutes at room temperature, split the blood equally between four 50-milliliter tubes and add 30 milliliters of sterile PBS supplemented with 1 millimolar EDTA to each tube. Mix again with a plastic 25-milliliter serological pipette and place the tubes in magnetic holders.

After 10 minutes, use a pipette to draw up the contents from the center of each tube, taking care not to aspirate more than 1 milliliter of red blood cells, and dispense the tube contents into one of four new 50-milliliter tubes. Add 500 additional microliters of the vortexed magnetic beads to each tube, and gently mix the cell solution. Place the tubes back into the magnetic holders for five minutes, before carefully transferring the contents from the center of each tube into one of four new 50-millimeter tubes.

When all of the cell suspensions have been transferred, place each new 50-milliliter tube into the magnet holders for five minutes before carefully transferring the tube contents into a third set of four new 50-milliliter tubes. Then, collect the cells by centrifugation, and resuspend all four cell pellets in a total of 10 milliliters of sterile PBS for counting.