Proliferative Assays for B Cell Function

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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.