Proliferative Assays for B Cell Function

James J. Mond1, Mark Brunswick1

1 Uniformed Services University of the Health Sciences, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.10
DOI:  10.1002/0471142735.im0310s57
Online Posting Date:  November, 2003
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Abstract

This unit describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen‐induced proliferation) requires the laborious procedures of isolating antigen‐specific B cells (which are otherwise present in too low a concentration in whole B cell populations). Cross‐linking of the B cell antigen receptor, surface immunoglobulin (sIg), by specific antigen stimulates cells to proliferate prior to secreting Ig. For this purpose, monoclonal or heterologous affinity‐purified anti‐Ig antibodies are used. B cells can also be stimulated to proliferate by antigen‐nonspecific reagents (mitogens), and it is also critical to study the role of these mitogens in B cell responses. Both of these systems have the advantage that the majority of B cells will be activated. The first basic protocol describes B cell proliferation induced by two commonly used stimulants–anti‐Ig antibody (either anti‐IgM or anti‐IgD) and lipopolysaccharide (LPS)–as measured by incorporation of [3H]thymidine into dividing cells. Alternate protocols describe other commonly used mitogens as well as other means of measuring cell proliferation.

     
 
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Table of Contents

  • Basic Protocol 1: ANTI‐IgM‐and LPS‐Stimulated B Cell Proliferation
  • Alternate Protocol 1: Alternate Stimuli for Polyclonal Activation of B Cells
  • Alternate Protocol 2: Quantitation of Cell Number Increase
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: ANTI‐IgM‐and LPS‐Stimulated B Cell Proliferation

  Materials
  • Purified resting B cells (units 3.5 & 3.8), prepared by Percoll gradient centrifugation
  • Complete DMEM‐10 medium, supplemented as in unit 3.8
  • Goat anti‐IgM (Jackson Immunoresearch) or bacterial LPS (E. coli; Difco #011B4)
  • 0.1 mCi/ml [3H]thymidine (20 Ci/mM; Du Pont NEN) in HBSS ( appendix 2A)
  • 15‐ml polypropylene tubes (Falcon #2059)
  • 96‐well flat‐bottom microtiter plates (Costar)
  • Harvesting system for collection of cells directly onto glass‐fiber strips (PHD Harvester, Cambridge Technologies)

Alternate Protocol 1: Alternate Stimuli for Polyclonal Activation of B Cells

  Additional Materials
  • 8‐MG (Sigma)
  • 0.1 N NaOH
  • 2 N HCl

Alternate Protocol 2: Quantitation of Cell Number Increase

  Additional Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.5 M EDTA prepared in PBS, ice‐cold
  • 95% ethanol, ice‐cold
  • 1 mM Hoechst 33342 (Sigma) prepared in H 2O
  • 1 mM Pyronin Y (Sigma) prepared in H 2O
  • 5‐ml round‐bottom centrifuge tubes (Falcon #2054)
  • Refrigerated low‐speed centrifuge (e.g., IEC 7R with 216 rotor)
  • Dual‐laser flow cytometer (e.g., FACScan, Becton Dickinson; unit 5.4)
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Figures

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Literature Cited

Literature Cited
   Abbas, A.K., Urioste, S., Collins, T.L., and Boom, W.H. 1990. Heterogeneity of helper/inducer T lymphocytes. IV. Stimulation of resting and activated B cells by Th1 and Th2 clones. J. Immunol. 144:2031‐2037.
   Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., and Crissman, H.A. 1987. Application of pyronin Y(G) in cytochemistry of nucleic acids. Cytometry. 8:138‐145.
   Diamantstein, T. and Blitstein‐Willinger, E. 1978. Specific binding of poly(I) poly(C) to the membrane of murine B lymphocyte subsets. Eur. J.Med. 8:896‐899.
   Feldbush, T.L. and Ballas, Z.K. 1985. Lymphokine‐like activity of 8‐mercaptoguanosine: Induction of T and B cell differentiation. J. Immunol. 134:3204‐3211.
   Goodman, M.G. and Weigle, W.O. 1983. Activation of lymphocytes by a thiol‐derivatized nucleoside: Characterization of cellular parameters and responsive populations. J. Immunol. 130:551‐558.
   Hodgkin, P.D., Yamashita, L.C., Coffman, R.L., and Kehry, M.R. 1990. Separation of events mediating B cell proliferation and Ig production using T cell membranes and lymphokines. J. Immunol. 145:2025‐2034.
   Mond, J.J., Balapure, A., Feuerstein, N., June, C.H., Brunswick, M., Lindsberg, M‐L., and Witherspoon, K. 1990. Protein kinase C activation in B cells by indolactam inhibits anti‐Ig‐mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation. J. Immunol. 144:451‐455.
   Puré, E. and Vitetta, E. 1980. Induction of murine B cell proliferation by insolubilized anti‐immunoglobulins. J. Immunol. 125:1240‐1242.
   Sieckmann, D.G., Asofsky, R., Mosier, D.E., Zitron, I.M., and Paul, W.E. 1978. Activation of mouse lymphocytes by anti‐immunoglobulin. I. Parameters of the proliferative response. J. Exp. Med. 147:814‐829.
   Swain, S.L., Howard, M., Kappler, J., Marrack, P., Watson, J., Booth, R., Wetzel, G.D., and Dutton, R.W. 1983. Evidence for two distinct classes of murine B cell growth factors with activities in different functional assays. J. Exp. Med. 158:822‐835.
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