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14/11/2014 (Added to site)
Author(s): Marjanovič, I.; Kandušer, M.; Miklavčič, D.; Keber, M. M.; Pavlin, M.

Comparison of Flow Cytometry, Fluorescence Microscopy and Spectrofluorometry for Analysis of Gene Electrotransfer Efficiency

Journal: Journal of Membrane Biology, 247/12 (2014), pp. 1259-1267
DOI: 10.1007/s00232-014-9714-4
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Abstract: In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.



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