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Proliferative Assays for B Cell Function

James J. Mond¹, Mark Brunswick¹

¹Uniformed 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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Unit Introduction
Basic Protocol: ANTI-IgM-and LPS-Stimulated B Cell Proliferation
Alternate Protocol: Alternate Stimuli for Polyclonal Activation of B Cells
Alternate Protocol: Quantitation of Cell Number Increase
Commentary
Literature Cited
Tables

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Materials

Basic Protocol: ANTI-IgM-and LPS-Stimulated B Cell Proliferation

Materials

Purified resting B cells (units 3.5A & 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: Alternate Stimuli for Polyclonal Activation of B Cells

Additional Materials

8-MG (Sigma)
0.1 N NaOH
2 N HCl

Alternate Protocol: 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 H₂O
1 mM Pyronin Y (Sigma) prepared in H₂O
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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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.