Animal Cell Separation and Subcellular Fractionation

Abstract

Cell populations isolated from tissues or from blood are remarkably complex containing many different types that can be dissociated and separated based on cell phenotype. Sorting and separation strategies differ widely as do the resulting cell populations in terms of both parameters upon which the sort is based and sample purity. Separation strategies can be simple or complex and based on culture characteristics or gene expression profiles. Each cell population itself contains many functionally different subcellular compartments that can also be selectively fractionated into subcellular components. Fractionation strategies must be tailored to provide the highest quality enrichment of the fraction of cells, organelles or complexes sought and after careful assessment of the target cell or molecule and the uses to which the final enriched fraction will be put.

Key Concepts:

  • Complex populations of cells from tissues or blood can be liberated and then sorted or separated for culture or subcellular fractionation.

  • Sorted cell populations can be fractionated into organelle compartments based on differential use of dilute detergents and differential centrifugation.

  • Sorted cell populations can be fractionated into functional subcellular compartments based on affinity of different molecules for the membrane and fibre systems within cells.

  • Careful evaluation of the purity and authenticity of each cell and molecular fraction is required before use or biological evaluation.

Keywords: cell separation; cell sorting; cell fractionation; cell compartments; organelles; mRNA populations; fibroblast depletion

Figure 1.

Multifluorescent channel‐based cell sorting. Cell sorting can be employed to facilitate the separation of discrete cell populations from complex mixed populations of cells based on cell surface markers that have been labelled with fluorochrome‐conjugated antibodies. Complex populations of peripheral blood mononuclear cells were labelled with four different antibodies each conjugated to a different fluorochrome. Colour back‐gating was applied to identify each cell population. Only the green population is positive for the green fluorochrome in the histogram (a). When two colours are analysed simultaneously, green and blue cells are positive for each of the two fluorochromes individually whereas red and purple cells are negative for both. Very rare magenta cells (circular gate) are positive for both green and blue fluorochromes (b). Each definable cell population, including very rare cells, can be individually sorted at high speed to physically isolate viable populations of cells.

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Further Reading

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Celis JE (2006) Cell Biology: A Laboratory Handbook, 3rd edn. San Diego: Academic Press.

Robinson LP, Darzynkiewicz Z, Hoffman R et al. (1997) Current Protocols in Cytometry. New York: Greene Publishing Association and Wiley Interscience.

Shapiro HM (2003) Practical Flow Cytometry, 4th edn. New York: Alan R Liss, Inc.

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Bird, R Curtis, Bird, Allison E Church, and DeInnocentes, Patricia(Jan 2011) Animal Cell Separation and Subcellular Fractionation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002588.pub2]