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Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun

Nature Protocols volume 1, pages 1517–1521 (2006)Cite this article

Abstract

Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h.

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Figure 1: Helios gene gun with the original accelerator chamber and associated spacer (boxed, and its replacement in the modified version).
Figure 2: Example of HEK293 cells shot with DiO/gold particles using the unmodified gene gun with gas pressure set at 120 psi.
Figure 3: Examples of neurons in mouse (a and b) and fish (c) cerebellum shot with DiO/gold particles using the modified gene gun with gas pressure at 50 psi.
Figure 4: Examples of fish Purkinje cells in cerebellum (Gnathonemus petersii) shot with DiO/gold particles using the modified gene gun with gas pressure at 50 psi.

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Acknowledgements

This work was supported by the Medical Research Council and the Wellcome Trust. S.C.R.L. holds a Wellcome Trust Senior Research Fellowship in Basic Biomedical Science.

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  1. Neurobiology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    John A O'Brien & Sarah C R Lummis

  2. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK

    Sarah C R Lummis

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  1. John A O'Brien
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Correspondence to Sarah C R Lummis.

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O'Brien, J., Lummis, S. Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun. Nat Protoc 1, 1517–1521 (2006). https://doi.org/10.1038/nprot.2006.258

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