Influence of a transiently transfected gene on apoptosis, measurements guided by cotransfected GFP

A rapid and quantitative flow cytometric method simultaneously identifying cells that incorporated a gene of interest in a transient electroporation and discriminating between dead, live and apoptotic states in a single measurement is presented. An expression vector encoding the gene of interest was cotransfected with a plasmid carrying the green fluorescent protein (GFP). Subsequently, the cultured cells were stained with 7-amino-actinomycin D (7-AAD) without fixation and were subjected to a multivariate analysis. The value of the method and its high reproducibility were demonstrated on Jurkat cells. Those cells were transiently transfected with a construct expressing a short C-terminal fragment of presenilin 1 (PS1-f) known to show anti-apoptotic properties. The PS1-f gene was under the control of the tetracycline-responsive transactivator.