This article describes a method for isolating monocytes from human whole blood using immunomagnetic negative selection. The process involves the use of antibody cocktails and magnetic beads to enrich the monocyte population for further analysis.
To isolate monocytes, a type of white blood cell, from whole blood by immunomagnetic negative selection, obtain anticoagulant-treated human blood in a tube. Add an antibody cocktail solution containing Fc receptor-blocking antibodies and bispecific tetrameric antibody complexes.
Each antibody complex comprises two monoclonal antibodies, with one antibody specific for cell surface antigens on the non-monocyte cells, including white and red blood cells, and the other antibody specific for dextran.
Add a dextran-coated magnetic bead solution. Incubate. The Fc receptor-blocking antibody binds to the monocytes' Fc receptor, preventing non-specific binding of monoclonal antibodies.
The monoclonal antibodies in antibody complexes recognize and bind to specific cell surface antigens on the non-monocyte cells, leaving the monocytes unlabeled. The dextran-coated magnetic beads bind to the antibody complexes' anti-dextran antibody, crosslinking the non-monocyte cells to the beads.
Place the tube in a magnetic holder. Under the influence of a high-gradient magnetic field, the magnetic bead-crosslinked cells bind to the tube walls, leaving the monocytes in the center of the tube.
Pipette the monocyte-containing solution; transfer to a fresh tube. Add magnetic beads, crosslinking remaining unwanted cells. Place under a magnetic field's influence, enriching the monocyte population.
Transfer the monocyte suspension to a fresh tube. Centrifuge the suspension. Resuspend the monocytes in a buffer for further analysis.
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.
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