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A simple and rapid nonviral approach to efficiently transfect primary tissue–derived cells using polyethylenimine

Nature Protocols volume 7, pages 935–945 (2012)Cite this article

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Abstract

This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.

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Figure 1
Figure 2: Effect of complexation volume on transfection efficiency.
Figure 3: Effect of PEI-to-pDNA weight ratios used in complex formation on transfection efficiency.
Figure 4: Effect of centrifugation and incubation time on transfection efficiency.

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  • 16 March 2016

     In the version of this article initially published, the amount of HEPES stated as being required to make up 100 ml of a 1 M stock solution was incorrect. Initially it was stated that 2.383 g was required; the correct amount is 23.83 g. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are grateful for financial support from the Natural Sciences and Engineering Research Council (NSERC) and Canadian Institutes of Health Research (CIHR). C.Y.M.H. is supported by the NSERC Alexander Graham Bell Canada Graduate Scholarship. We thank C. Kucharski for the isolation and cell culturing of rat bone marrow stromal cells.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

    Charlie Yu Ming Hsu & Hasan Uludağ

  2. Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alberta, Canada

    Hasan Uludağ

  3. Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

    Hasan Uludağ

Authors
  1. Charlie Yu Ming Hsu
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Contributions

C.Y.M.H. wrote the manuscript. C.Y.M.H. and H.U. undertook the editing and revision of the manuscript during the publication process. C.Y.M.H. did the experimental work. C.Y.M.H. and H.U. designed the experiments and analyzed the experimental data.

Corresponding author

Correspondence to Hasan Uludağ.

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The authors declare no competing financial interests.

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Hsu, C., Uludağ, H. A simple and rapid nonviral approach to efficiently transfect primary tissue–derived cells using polyethylenimine. Nat Protoc 7, 935–945 (2012). https://doi.org/10.1038/nprot.2012.038

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