Clonal Isolation and Plating Efficiency

Abstract

Viable cultures of cells that were initiated from single cells can be obtained. However, both the isolation of the single cell of interest and the ability to promote proliferation of this cell present challenges. Selection of the single cell can be achieved in a number of ways. Among the methods discussed are a direct approach (cloning cylinders or discs), a theoretical approach (limiting dilution) and isolation using cell sorters. Any of these approaches can be combined with a pre‐seeding technique that will enrich the population of the desired cells in the culture. A number of techniques to address the second challenge, cell proliferation, are also addressed. These include the use of culture ware with a small growth area, increasing the likelihood of cell attachment, the enrichment of the culture medium and the use of feeder layer cells. Because cells differ widely in their characteristics, a combination of techniques can be used.

Key Concepts:

  • Cloned cells in culture are derived from a single cell.

  • Each of the cells in a clone should have identical characteristics.

  • Selection of cells for cloning may be aided by fluorescent molecules and FACS.

  • The survival and proliferation of the cells that form clones require special conditions.

Keywords: adherent cells; single‐cell isolation; single‐cell survival; cloning cylinders and discs; FACS

Figure 1.

Single‐cell isolation using a cloning cylinder. (a) The cell of interest (triangular) is surrounded by the dotted line. (b) The cloning cylinder is placed over the cell. (c) A solution to release the cell is added inside the cloning cylinder using a pipette tip. (d) When released, the cell is placed in a small well. Modified from Martin (, p. 146).

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References

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Martin BM (2011) unpublished results.

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Further Reading

Aside from journal articles, books and monographs, trade journals and trusted vendors are excellent sources of current advancements and background information. Examples of such are: Biotechniques.com: http://www.biotechniques.com

Freshney RI (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edn. Hoboken: John Wiley & Sons.

Freshney RI, Stacey GN and Auerbach JM (eds) (2007) Culture of Human Stem Cells. Hoboken: John Wiley & Sons.

Sigma‐Aldrich Informational Publications: http://www.sigma‐aldrich.com or http://www.sigma‐aldrich.com/techservice

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How to Cite close
Martin, Bernice M(Dec 2011) Clonal Isolation and Plating Efficiency. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002557.pub2]