Isolation of Ly‐1+/CD5+ B Cells by Cell Sorting

Richard R. Hardy1

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.5B
DOI:  10.1002/0471142735.im0305bs55
Online Posting Date:  August, 2003
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Ly‐1/CD5 is a 68‐kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells. Although its function remains the subject of speculation, CD5 expression serves as a useful marker for a functionally distinct population of B cells that has attracted a considerable amount of interest from investigators of both murine and human immune systems. Of critical importance in much of this work is the isolation of pure populations of CD5+ B cells along with appropriate control populations. A flow cytometry technique is presented in this unit, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: CD5+ B Cell Enrichment Using Cell Sorting
  • Alternate Protocol 1: Selective Depletion of B Cell Subsets by Cell Sorting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: CD5+ B Cell Enrichment Using Cell Sorting

  • Mouse (one or more, depending on cell number requirements)
  • Staining medium, ice cold
  • 0.165 M NH 4Cl lysis solution, ice cold
  • Fetal or newborn calf serum
  • 0.4% trypan blue (store in dark bottle and filter after prolonged storage; GIBCO/BRL)
  • Staining reagents: monoclonal anti‐CD5, anti‐IgM, or anti‐IgD antibodies and appropriate control fluorescent reagents (Tables 3.5.1 & 3.5.2)
  • Second‐step antibody reagent (optional; see Table 3.5.2)
  • 10× propidium iodide solution
  • Supplemented RPMI 1640 medium
  • 70% ethanol or dilute detergent (e.g., Linbro 7×)
  • Iris forceps
  • 10‐ml syringes equipped with 25‐G, ⅝‐in. needles
  • 1‐in. nylon mesh squares (cut from 40‐in. rolls of 41‐µM‐pore Nitex; Tetko)
  • 15‐ml polypropylene conical centrifuge tube
  • Frosted glass microscope slides
  • Low‐speed refrigerated centrifuge (e.g., Beckman GPKR equipped with GH‐3.7 swinging‐bucket rotor)
  • Hemacytometer
  • 0.22‐µm filter (Millex‐GV4, Millipore)
  • Flow cytometer (units 5.1 & 5.4)
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), preparation of peritoneal feeder cells (unit 3.15), trypan blue exclusion ( appendix 3A), and flow cytometry (units 5.1 5.4)
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Literature Cited

   Coffman, R.L. 1982. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre‐B cell development. Immunol. Rev. 69:5‐23.
   Coffman, R.L. and Weissman, I.L. 1981. A monoclonal antibody that recognizes B cells and B cell precursors in mice. J. Exp. Med. 153:269‐279.
   Hardy, R.R. and Hayakawa, K. 1986. Development and physiology of Ly‐1 B and its human homolog, Leu‐1 B. Immunol. Rev. 93:53‐79.
   Hayakawa, K. and Hardy, R.R. 1988. Normal, autoimmune, and malignant CD5+ B cells: The Ly‐1 B lineage? Annu. Rev. Immunol. 6:197‐218.
   Hayakawa, K., Hardy, R.R., Parks, D.R., and Herzenberg, L.A. 1983. The “Ly‐1 B” cell subpopulation in normal, immunodefective, and autoimmune mice. J. Exp. Med. 157:202‐218.
   Herzenberg, L.A., Stall, A.M., Lalor, P.A., Sidman, C., Moore, W.A., Parks, D.R., and Herzenberg, L.A. 1986. The Ly‐1 B cell lineage. Immunol. Rev. 93:81‐102.
   Hillson, J.L., Oppliger, I.R., Sasso, E.H., Milner, E.C., and Wener, M.H. 1992. Emerging human B cell repertoire. Influence of developmental stage and interindividual variation. J. Immunol. 149:3741‐3752.
   Ho, M.K. and Springer, T.A. 1982. Mac‐1 antigen: Quantitative expression in macrophage populations and tissues, and immunofluorescent localization in spleen. J. Immunol. 128:2281‐2286.
   Kincade, P.W., Lee, G., Sun, L., and Watanabe, T. 1982. Monoclonal rat antibodies to murine IgM determinants. J. Immunol. Methods. 42:17‐26.
   Kipps, T.J. 1989. The CD5 B Cell. Adv. Immunol. 47:117‐185.
   Ledbetter, J.A. and Herzenberg, L.A. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63‐90.
   Li, Y., Hayakawa, K., and Hardy, R.R. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178:951‐960.
   Oi, V.T., Glazer, A.N., and Stryer, L. 1982. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J. Cell Biol. 93:981‐986.
   Parks, D.R. and Herzenberg, L.A. 1984. Fluorescence‐activated cell sorting: Theory, experimental optimization, and applications in lymphoid cell biology. Methods Enzymol. 108:197‐241.
   Rao, M., Lee, W.T., and Conrad, D.H. 1987. Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE. J. Immunol. 138:1845‐1851.
   Schuppel, R., Wilke, J., and Weiler, E. 1987. Monoclonal anti‐allotype antibody towards BALB/c IgM. Analysis of specificity and site of a V‐C crossover in recombinant strain BALB‐Igh‐Va/Igh‐Cb. Eur. J. Immunol. 17:739‐741.
   Stall, A.M. and Loken, M.R. 1984. Allotypic specificities of murine IgD and IgM recognized by monoclonal antibodies. J. Immunol. 132:787‐795.
   Waldschmidt, T.J., Kroese, F.G., Tygrett, L.T., Conrad, D.H., and Lynch, R.G. 1991. The expression of B cell surface receptors. III. The murine low‐affinity IgE Fc receptor is not expressed on Ly 1 or ‘Ly 1‐like, B cells’. Int. Immunol. 3:305‐315.
Key References
   Carmack, C.E., Shinton, S.A., Hayakawa, K., and Hardy, R.R. 1990. Rearrangement and selection of VH11 in the Ly‐1 B cell lineage. J. Exp. Med. 172:371‐374.
  These papers all describe analyses of fractionated populations of CD5 B cells and control populations.
   Hardy, R.R., Carmack, C.E., Shinton, S.A., Riblet, R.J., and Hayakawa, K. 1989. A single VH gene is utilized predominantly in anti‐BrMRBC hybridomas derived from purified Ly‐1 B cells. Definition of the VH11 family. J. Immunol. 142:3643‐3651.
   Hayakawa, K., Hardy, R.R., and Herzenberg, L.A. 1986. Peritoneal Ly‐1 B cells: Genetic control, autoantibody production, increased lambda light chain expression. Eur. J. Immunol. 16:450‐456.
   Hayakawa, K., Carmack, C.E., Hyman, R., and Hardy, R.R. 1990. Natural autoantibodies to thymocytes: Origin, VH genes, fine specificities, and the role of Thy‐1 glycoprotein. J. Exp. Med. 172:869‐878.
   Hayakawa, K., Hardy, R.R., Honda, M., Herzenberg, L.A., Steinberg, A.D., and Herzenberg, L.A. 1984. Ly‐1 B cells: Functionally distinct lymphocytes that secrete IgM autoantibodies. Proc. Natl. Acad. Sci. U.S.A. 81:2494‐2498.
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