Purification of transiently transfected cells by magnetic affinity cell sorting |
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Authors: | Raji Padmanabhan Christopher D Corsico Tazuko H Howard Wolfgang Holter C Michael Fordis Mark Willingham Bruce H Howard |
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Institution: | Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892. |
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Abstract: | A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained. |
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Keywords: | recombinant technology cell culture techniques cell sorter receptors general DNA gene expression |
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