High gradient magnetic separation of rosette-forming cells

Abstract

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (less than 2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20-30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20-50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90-100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80-90%.