Direct Introduction of Molecules into Cells

Paul L. McNeil1

1 Medical College of Georgia, Augusta, Georgia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.1
DOI:  10.1002/0471143030.cb2001s18
Online Posting Date:  May, 2001
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Abstract

Techniques for introducing normally impermeant macromolecules into the living cell—referred to as “cell‐loading techniques”—are useful in a variety of settings for the cell biologist. Microinjection is probably the most commonly used technique for introducing fluorescent probes, fluorescently tagged proteins, and antibodies into living cells for short‐term studies of cell physiology and protein location and function. It is, however, not the only technique available, nor the easiest or least expensive to implement. Among the alternatives are several closely related techniques that, like microinjection, rely on the cell's ability to reseal a mechanically induced plasma membrane disruption created in order to gain temporary access to cell cytosol. Four such techniques are described in this unit: scrape loading, scratch loading, bead loading, and syringe loading. Unlike microinjection, these techniques allow one to rapidly load (in a matter of minutes) thousands or even many millions of many types of mammalian cells with normally impermeant molecules, and so to facilitate quantitative analyses of the effect of loading.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Scrape Loading
  • Alternate Protocol 1: Scratch Loading
  • Alternate Protocol 2: Bead Loading
  • Alternate Protocol 3: Syringe Loading
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Scrape Loading

  Materials
  • Adherent cells of interest, growing in tissue culture (also see unit 1.1)
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A) or equivalent physiological saline containing 1 to 1.5 mM CaCl 2 at physiological temperature (37°C for mammalian cells)
  • Loading solution: DPBS (with 1 to 1.5 mM calcium) containing molecule to be loaded, at physiological temperature (37°C for mammalian cells)
  • Rubber policeman
  • Circular tissue culture dishes
  • Additional reagents and equipment for cell culture (unit 1.1)

Alternate Protocol 1: Scratch Loading

  • 30‐G syringe needle or similar sharp implement (e.g., Fisher)
  • Glass coverslips

Alternate Protocol 2: Bead Loading

  • Glass beads, 50‐ to 500‐µM diameter (Sigma)
  • Glass coverslips

Alternate Protocol 3: Syringe Loading

  • Adherent or suspension cells of interest, growing in tissue culture (unit 1.1)
  • 30‐G syringe needle and 1‐ml syringe or micro‐ or ultramicropipettor (1‐ to 10‐µl range) and appropriate pipet tips (e.g., Fisher)
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Figures

Videos

Literature Cited

Literature Cited
   Clarke, M.S.F. and McNeil, P.L. 1992. Syringe loading introduces macromolecules into living mammalian cell cytosol. J. Cell Sci. 102:535‐541.
   Clarke, M.S.F. and McNeil, P.L. 1994. Syringe loading: A method for inserting macromolecules into cells in suspension. In Cell Biology: A Laboratory Handbook, vol. 3. (J.E. Celis, ed.) pp. 30‐36. Academic Press, San Diego, Calif.
   Doberstein, S.K., Baines, I.C., Wiegand, G., Korn, E.D., and Pollard, T.D. 1993. Inhibition of contractile vacuole function in vivo by antibodies against myosin‐I [see comments]. Nature . 365:841‐843.
   McNeil, P.L. 2002. Repairing a torn cell surface: Make way, lysosomes to the rescue. J. Cell Sci. 115:873‐879.
   McNeil, P.L. and Steinhardt, R.A. 1997. Loss, restoration and maintenance of plasma membrane integrity. J. Cell Biol. 137:1‐4.
   McNeil, P.L. and Warder, E. 1987. Glass beads load macromolecules into living cells. J. Cell Sci. 88:669‐678.
   McNeil, P.L., Murphy, R.F., Lanni, F., and Taylor, D.L. 1984. A method for incorporating macromolecules into adherent cells. J. Cell Biol . 98:1556‐1564.
   Swanson, J.A. and McNeil, P.L. 1987. Nuclear reassembly excludes large macromolecules. Science 238:548‐550.
   Terasaki, M., Miyake, K., and McNeil, P.L. 1997. Large plasma membrane disruptions are rapidly resealed by Ca2+‐dependent vesicle‐vesicle fusion events. J. Cell Biol. 139:63‐74.
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