Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells
Egea Navarro, Joaquim
Comella i Carnicé, Joan Xavier
MetadataShow full item record
One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones. In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with vectors 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 detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequate 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 interest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells. Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.
Is part ofJournal of Neuroscience Methods, 2000, vol. 100, núm. 1-2, p. 63-69
European research projects
Showing items related by title, author, creator and subject.
Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells Egea Navarro, Joaquim; Espinet Mestre, Carme; Comella i Carnicé, Joan Xavier (Wiley, 1998)In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes ...
Calcium Influx Activates Extracellular-regulated Kinase/Mitogen-activated Protein Kinase Pathway through a Calmodulin-sensitive Mechanism in PC12 Cells Egea Navarro, Joaquim; Espinet Mestre, Carme; Comella i Carnicé, Joan Xavier (American Society for Biochemistry and Molecular Biology, 1999)Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the ...
Egea Navarro, Joaquim; Espinet Mestre, Carme; Soler i Tatché, Rosa Ma.; Dolcet Roca, Xavier; Yuste Mateos, Víctor J. (Víctor José); Encinas Martín, Mario; Iglesias Durán, Montserrat; Rocamora Ibars, Nativitat; Comella i Carnicé, Joan Xavier (Rockefeller University Press, 2001)It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, ...