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.
Vézina
J
J
Preclinical Research, Novartis Pharma Ltd., CH-4002, Basel, Switzerland. jvezina@oci.utoronto.ca
Grossmüller
F
F
Müller
K
K
D017209:Apoptosis; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D053764:Presenilin-1; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D019014:fas Receptor; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; C025942:7-aminoactinomycin D