Cell Separation Techniques Used in Immunology

Abstract

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.)

close

References

Altman JD, Moss PAH, Goulder PJR et al. (1996) Phenotypic analysis of antigen‐specific T lymphocytes. Science 274: 94–96.

Böyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scandinavian Journal of Clinical Laboratory Investigation, Supplement 97: 77–89.

Lalor PA, Nossal GJ, Sanderson RD and McHeyzer Williams MG (1992) Functional and molecular characterization of single, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP)‐specific, IgG1+ B cells from antibody‐secreting and memory B cell pathways in the C57BL/6 immune response to NP. European Journal of Immunology 22: 3001–3011.

Mason DW and Williams AF (1986) Kinetics of antibody reactions and the analysis of cell surface antigens. In: Weir DM, Herzenberg LA, Blackwell C and Herzenberg LA (eds) Handbook of Experimental Immunology, vol. 1, 4th edn, pp. 38.1–38.17. Oxford: Blackwell Scientific.

Reif AE (1989) The discovery of Thy‐1 and its development. In: Reif AE and Schlesinger M (eds) Cell Surface Antigen Thy‐1, vol. 45, pp. 3–45. New York: Marcel Dekker.

Rembaum A and Dreyer WJ (1980) Immunomicrospheres: reagents for cell labeling and separation. Science 208: 364–368.

Scheffold A, Miltenyi S and Radbruch A (1995) Magnetofluorescent liposomes for increased sensitivity of immunofluorescence. Immunotechnology 1: 127–137.

Seed B (1995) Developments in expression cloning. Current Opinion in Biotechnology 6: 567–573.

Wysocki LJ and Sato VL (1978) ‘Panning’ for lymphocytes: a method for cell selection. Proceedings of the National Academy of Sciences of the USA 75: 2844–2848.

Zlokarnik G, Negulescu PA, Knapp TE et al. (1998) Quantitation of transcription and clonal selection of single living cells with beta‐lactamase as reporter. Science 279: 84–88.

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.

Kaufmann HE and Kabelitz D (eds) (1998) Immunology of infection. In: Methods in Microbiology, vol. 25. San Diego: Academic Press.

Klaus GGB (ed.) (1987) Lymphocytes; a Practical Approach. Oxford: IRL Press.

Lis H and Sharon N (1998) Lectins: carbohydrate‐specific proteins that mediate cellular recognition. Chemical Reviews 98: 637–674.

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.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
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]