Ballistic Labeling with Fluorescent Dyes and Indicators

Joshua L. Morgan1, Rachel O.L. Wong1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.11
DOI:  10.1002/0471142301.ns0211s43
Online Posting Date:  April, 2008
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Abstract

Neuronal cell labeling is fundamental to investigations of the nervous system. Labeling of cells in live or fixed tissue with dyes or ion indicators using ballistic approaches has recently been developed for the study of neuronal architecture and function. In this approach, dye‐coated particles are propelled into cells by a pulse of pressurized helium. This unit provides step‐by‐step protocols for coating tungsten particles with fluorescent or indicator dyes and for delivering these particles into cells and tissue. The major advantage of the ballistic method of dye delivery is that large populations of neurons can be rapidly labeled within a piece of live or fixed tissue. Advantages and limitations of the approach are discussed and technical advice is provided. Curr. Protoc. Neurosci. 43:2.11.1‐2.11.10. © 2008 by John Wiley & Sons, Inc.

Keywords: ballistic cell‐labeling; Gene Gun; particle‐mediated dye delivery; fluorescent dyes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Dye‐Coated Particles for Ballistic Delivery into Cells
  • Basic Protocol 2: Ballistic Delivery of Dye‐Coated Particles into Cells or Tissue
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Dye‐Coated Particles for Ballistic Delivery into Cells

  Materials
  • Dextran‐conjugated dyes and ion indicators (Molecular Probes):
    • Fluorescent dextrans (e.g., 10,000 MW; Oregon green 488, dextran‐conjugated Alexa dyes)
    • Calcium indicators conjugated to dextrans (e.g., Fura‐2 dextran, Oregon Green 488 BAPTA‐1, Calcium Green dextran)
  • Double‐distilled water
  • 1.0‐ to 1.7‐µm tungsten particles (Bio‐Rad)
  • 70% (v/v) ethanol
  • 20 mg/ml polyvinylpyrrolidone (PVP) in 100% ethanol; store in plastic tubes up to 1 month at room temperature
  • Carbocyanine dyes (Molecular Probes):
    • Octadecyl (C18) indocarbocyanines (DiI and DiD)
    • Oxacarbocyanine (DiO)
  • Methylene chloride (e.g., Sigma)
  • 1.6‐ml microcentrifuge tube with locking snap cap
  • Glass slide (any size)
  • Single‐edge razor blade
  • Wax weighing paper
  • Tefzel tubing (Bio‐Rad)
  • Parafilm
  • Helios Gene Gun guillotine and coating station (Bio‐Rad)
  • Kimwipe or tissue paper
  • Capped jar or scintillation vial
  • Dessicant (for bullet storage), enclosed in gel capsule
  • 5‐ or 10‐ml syringe
  • 15‐ml conical plastic tubes
  • Sonicator
  • Nitrogen gas tanks
  • Aluminum foil

Basic Protocol 2: Ballistic Delivery of Dye‐Coated Particles into Cells or Tissue

  Materials
  • Tissue sample
  • Bullets with dye‐coated particles ( protocol 1)
  • Culture medium or buffer appropriate for sample
  • Buffer (e.g., phosphate‐buffered saline, PBS; appendix 2A), optional
  • Nail polish, optional
  • Helios Gene Gun with barrel barrel liner for plasmid shooting (Bio‐Rad)
  • 3.0‐µm pore size, high density, 6‐well format tissue culture filter inserts (e.g., Falcon)
  • Helium cartridge with low pressure helium regulator (e.g., 0 to 200 psi range)
  • Recording chamber (for live tissue) or glass slide and coverslip
  • Parafilm, optional
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Figures

Videos

Literature Cited

Literature Cited
   Benediktsson, A.M., Schachtele, S.J., Green, S.H., and Dailey, M.E. 2005. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J. Neurosci. Methods 141: 41‐53.
   Bittman, K.S., Panzer, J.A., and Balice‐Gordon, R.J. 2004. Patterns of cell‐cell coupling in embryonic spinal cord studied via ballistic delivery of gap‐junction‐permeable dyes. J. Comp. Neurol. 477: 273‐285.
   Connaughton, V.P., Graham, D., and Nelson, R. 2004. Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina. J. Comp. Neurol. 477: 371‐385.
   Gan, W.B., Grutzendler, J., Wong, W.T., Wong, R.O., and Lichtman, J.W. 2000. Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27: 219‐225.
   Grutzendler, J., Tsai, J., and Gan, W.B. 2003. Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes. Methods 30: 79‐85.
   Haas, K., Sin, W.C., Javaherian, A., Li, Z., and Cline, H.T. 2001. Single‐cell electroporation for gene transfer in vivo. Neuron 29: 583‐591.
   Kettunen, P., Demas, J., Lohmann, C., Kasthuri, N., Gong, Y., Wong, R.O., and Gan, W.B. 2002. Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators. J. Neurosci. Methods 119: 37‐43.
   Lang, S.B., Bonhoeffer, T., and Lohmann, C. 2006. Simultaneous imaging of morphological plasticity and calcium dynamics in dendrites. Nat. Protoc. 1: 1859‐1864.
   Lo, D.C., McAllister, A.K., and Katz, L.C. 1994. Neuronal transfection in brain slices using particle‐mediated gene transfer. Neuron 13: 1263–1268.
   Matsuda, T. and Cepko, C.L. 2004. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101: 16‐22.
   Moolman, D.L., Vitolo, O.V., Vonsattel, J.P., and Shelanski, M.L. 2004. Dendrite and dendritic spine alterations in Alzheimer models. J. Neurocytol. 33: 377‐387.
   O'Brien, J. and Lummis, S.C. 2004. Biolistic and diolistic transfection: Using the Gene Gun to deliver DNA and lipophilic dyes into mammalian cells. Methods 33: 121‐125.
   O'Brien, J.A. and Lummis, S.C. 2006. Diolistic labeling of neuronal cultures and intact tissue using a hand‐held Gene Gun. Nat. Protoc. 1: 1517‐1521.
   Qin, Y., Xu, G., and Wang, W. 2006. Dendritic abnormalities in retinal ganglion cells of three‐month diabetic rats. Curr. Eye Res. 31: 967‐974.
   Sun, W., Li, N., and He, S. 2002. Large‐scale morphological survey of rat retinal ganglion cells. Vis. Neurosci. 19: 483‐493.
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