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Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Nature Protocols volume 5, pages 1993–2004 (2010)Cite this article

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Abstract

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19+ Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.

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Figure 1: Selection scheme based on FACS.
Figure 2: Workflow for setting up and executing a FACS-SELEX experiment.
Figure 3: Monitoring enrichment of ssDNA aptamers during FACS-SELEX.
Figure 4: Determination of the relative amount of ssDNA library bound to Burkitt's lymphoma cells in the absence (w/o) and presence of increasing amounts of competitor (salmon sperm DNA).

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Acknowledgements

This research was supported by research grants from the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 704, the Graduiertenkolleg GRK 804 and the Fonds der Chemischen Industrie. We thank N. Krämer for excellent technical assistance and J. Müller for support during the preparation of peripheral blood mononuclear cells from whole blood.

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Authors and Affiliations

  1. Department of Life and Medical Sciences, University of Bonn, Bonn, Germany

    Günter Mayer, Marie-Sophie L Ahmed & Michael Famulok

  2. Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany

    Andreas Dolf, Elmar Endl & Percy A Knolle

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  1. Günter Mayer
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Contributions

G.M. and M.-S.L.A., along with M.F., performed and designed most of the included studies. E.E. and A.D. provided experimental input in setting up the FACS experiments and performed some of them to establish optimal conditions, assisted by M.-S.L.A. and G.M. P.A.K. provided conceptual input. M.F. and G.M. supervised the research project and assisted in the experimental design. All authors discussed the experimental results. M.F. and G.M. wrote the manuscript.

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Correspondence to Günter Mayer or Michael Famulok.

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The authors declare no competing financial interests.

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Mayer, G., Ahmed, MS., Dolf, A. et al. Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat Protoc 5, 1993–2004 (2010). https://doi.org/10.1038/nprot.2010.163

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