Cell Separation Techniques Used in Immunology


General methods for separating subpopulations of cells involved in immune responses include those based on differences in cell density, surface adherence, and the presence or absence of characteristic cell surface molecules, especially those recognized by CD monoclonal antibodies. The choice of technique depends on the scale of the separation; the trade‐offs between starting fraction, purity and yield; and the sophistication and cost of reagents and apparatus. Licensing for clinical use may impose constraints on a method's suitability.

Keywords: cell density; antigen‐specific cells; cell adherence; sedimentation; flow cytometry

Figure 1.

Principles of approach to separation. In positive selection, a marker that identifies the required subset is picked up by the separation method. In negative selection, a marker on the complementary subset is used, and the cells that are left behind are the wanted ones. The net purification result is similar by either method, except that with positive selection the marker has been exposed to the antibody or ligand that recognizes it.

Figure 2.

These FACS analytical profiles of rat lymph node cells stained with three different monoclonal antibodies illustrate three situations of labelling. In (a) (CD4) there is a very sharp homogeneous peak of brightly stained cells with a long trough separating them from background. In (b) (stained with anti‐IgD) the distribution is more heterogeneous, and there is a less clear demarcation between labelled and unlabelled. In (c) (HIS22) the peaks are nearly merged, making separation much more difficult. (Data obtained by Wendy Brownsill and Margaret Hughes.)



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Further Reading

Barclay AN, Brown MH, Law SK, McKnight AJ, Tomlinson MG and van der Merwe PA (eds) (1997) The Leukocyte Antigen Factbook, 2nd edn. San Diego: Academic Press.

Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W (eds) (1997) Current Protocols in Immunology. New York: Wiley.

Herzenberg LA, Weir DM, Herzenberg LA and Blackwell C (eds) (1997) Handbook of Experimental Immunology, 5th edn. Oxford: Blackwell Scientific.

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McKnight AJ and Gordon S (1998) Membrane molecules as differentiation antigens of murine macrophages. Advances in Immunology 68: 271–314.

Recktenwald D and Radbruch A (eds) (1998) Cell Separation Methods and Applications. New York: Marcel Dekker.

Rose NR, de Macario EC, Folds JD, Lane HC and Nakamura RM (eds) (1997) Manual of Clinical Laboratory Immunology, 5th edn. Oxford: Blackwell Scientific.

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Hunt, Simon V(Apr 2001) Cell Separation Techniques Used in Immunology. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0001122]