Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection
Transkript
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.
