Unwanted cells are removed by aspiration while the tube remains in the magnet. Magnetically tagged cells will migrate toward the tube wall on the magnet side (desired cell population), leaving the untagged (unwanted) cells in suspension. The tube containing the cell suspension is placed in the magnet. The cells are tagged with an anti-CD25 biotinylated antibody followed by addition of streptavidin ferrofluid. In Step 2, the negatively isolated CD4 + T cells from Step 1 are subjected to positive selection. Magnetically tagged cells (unwanted cell fraction) will migrate toward the magnet, leaving the untagged cells (desired cell population) in suspension to be harvested by aspiration while the tube remains in the magnetic field. The tube containing the cell suspension is then placed within a magnetic field. Streptavidin ferrofluid is added to the reaction, and the streptavidin coated nanoparticles interact with the biotinylated antibody tagged cells. In Step 1, a mononuclear cell suspension is incubated with a biotinylated antibody cocktail which targets unwanted cells.
Isolation of CD4 + CD25 + regulatory T cells is performed using a combination of negative and positive selection methods. This protocol provides a basic guide for the magnetic selection of CD4 + CD25 + Regulatory T lymphocytes from preparations of peripheral blood mononuclear cells (PBMC) or splenocytes.
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