Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells

One of the more time-consuming procedures in the study of exogenously expre ssed proteins in cell lines is the selection of individual transfected clon es. In recent yeats, green fluorescent protein variants with excitation/emi ssion spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with ve ctors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter. In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detecti on of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequa te combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of i nterest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiv eness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein ki nase pathway, resulting in the spontaneous differentiation of PC12 cells. A lso, this procedure has been used with other set of expression vectors enco ding proteins that protect PC12 cells from apoptosis caused by different st imuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 c ells. (C) 2000 Elsevier Science B.V. All rights reserved.