EFFICIENT IMMUNOSELECTION OF CYTOLYTIC EFFECTORS WITH A MAGNETIC CELL SORTER

This paper describes a rapid and efficient method for the sorting of in vitro activated cytolytic effectors cells. For cytotoxic assays, a large number of cells with conserved function must be rapidly obtained. Immunomagnetic sorting was chosen because it is faster than flow cytometry sorting. The MACS system requires the use of paramagnetic beads of small diameter (100-150 nm), reputed to interfere minimally with cell function. In order to generate the cytolytic effectors, peripheral blood lymphocytes were cultivated in the presence of interleukin-2 (50 U/ml) and anti-CD3 monoclonal antibody (BMA030, 100 ng/ml) for 4 days. Cell separation was based on the membrane expression of the CD3 complex. The purity obtained for positive (CD3+) cell sorting with the MACS was higher than 95 %. The purity of negative (CD3-) cell fraction was more variable, but further purification by flow cytometry rapidly yielded purity higher than 95 %. Cytotoxic assays were performed against four target cell lines (K562, Daudi, HL60 and U937) and proliferation assays showed that both negatively and positively selected populations had conserved their function acquired during culture in the presence of anti-CD3 mAb and IL2.