Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation.
A method for labelling the intracellular ras oncogene product, p21, with a monoclonal antibody, in B16BL6 mouse melanoma cells for subsequent flow cytometric analysis and viable cell sorting is described. Permeabilization of the cells for introduction of labelled antibody was attempted using (1) lysolecithin treatment, and (2) electroporation, a much more highly controllable technique. Permeabilization was assessed using propidium iodide or calcofluor white M2R staining, while short-term cellular viability was determined using fluorescein diacetate staining and long-term viability by reculturing the sorted cells. We successfully introduced labelled antibody into the cells with both permeabilization techniques. Insufficient numbers of viable permeabilized cells were obtained lysolecithin treatment to warrant an attempt at viable cell sorting. On the other hand, good numbers of viable, permeabilized cells were obtained using electroporation and we successfully sorted viable tumor cell populations based on the intensity of their anti-p21ras staining. These sorted tumor cells retained their characteristic anti-p21ras staining intensity for at least 2 weeks of propagation in culture.
Berglund
D L
DL
Department of Biology, Montana State University, Bozeman 59717.
Starkey
J R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002463:Cell Membrane Permeability; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002999:Clone Cells; D004560:Electricity; D005434:Flow Cytometry; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015689:Oncogene Protein p21(ras)