A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry.
In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.
Ogris
M
M
Institute of Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030, Vienna, Austria.
Wagner
E
E
Steinlein
P
P
D002448:Cell Adhesion; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006801:Humans; D020014:K562 Cells; D011073:Polyamines; D000071228:Polyelectrolytes; D013050:Spectrometry, Fluorescence
D004247:DNA; D005456:Fluorescent Dyes; D011073:Polyamines; D000071228:Polyelectrolytes