Purification of transiently transfected cells by magnetic affinity cell sorting.
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.
Padmanabhan
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Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Corsico
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Howard
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Holter
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Fordis
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Willingham
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Howard
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D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008280:Magnetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D011971:Receptors, Immunologic / Q000235:genetics; D015375:Receptors, Interleukin-2; D014158:Transcription, Genetic; D014162:Transfection; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; D014764:Viral Proteins / Q000235:genetics
D000954:Antigens, Surface; D004274:DNA, Recombinant; C029152:G-substrate; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; D014764:Viral Proteins