Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells
Issue date
2000Author
Egea Navarro, Joaquim
Comella i Carnicé, Joan Xavier
Suggested citation
Espinet Mestre, Carme;
Gómez Arbonés, Javier;
Egea Navarro, Joaquim;
Comella i Carnicé, Joan Xavier;
.
(2000)
.
Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells.
Journal of Neuroscience Methods, 2000, vol. 100, núm. 1-2, p. 63-69.
https://doi.org/10.1016/S0165-0270(00)00233-8.
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Show full item recordAbstract
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.