A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures

Pike, Beverley L.; Jennings, Gael; Shortman, Ken

doi:10.1016/0022-1759(82)90346-5

Cited by 12 publications

(5 citation statements)

References 16 publications

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“…PFC Assay. We performed reverse PFC assays in 96-well microtiter plates (Linbro Chemical Co., Hamden, CT) by the method of Pike et al (32). We prepared protein A-…”

Section: Methodsmentioning

confidence: 99%

“…Such SN contain B cell growth factor (BCGF), B cell differentiation factor for IgM (BCDFu) and IgG (BCDF3"), colony-stimulating factors, and histamineproducing-cell-stimulating factor (HCSF or interleukin 3 [IL-3]), but not 3"-interferon, or conventional (30) T cell-replacing factor (TRF) (16,29,31).PFC Assay. We performed reverse PFC assays in 96-well microtiter plates (Linbro Chemical Co., Hamden, CT) by the method of Pike et al (32). We prepared protein A-…”

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confidence: 99%

See 1 more Smart Citation

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

Layton¹,

Vitetta²,

Uhr³

et al. 1984

The Journal of Experimental Medicine

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The switch from IgM to IgG (or IgA, IgE) synthesis has been studied in both normal and neoplastic Ig-secreting cells (1-6). Evidence suggests that DNA rearrangements precede isotype switching. The nature of the signals that induce isotype switching are not understood, although T cells (7-13), T cell-derived lymphokines (14)(15)(16)(17)(18)(19)(20), and mitogens (20-22) can influence switching in activated B cells.We have previously (16) described a T cell-derived lymphokine termed "B cell differentiation factor for IgG" (BCDF3,), l that induces increased levels of IgG1 secretion in lipopolysaccharide (LPS)-stimulated splenic B cells. T cell supernatants (SN) containing BCDF3, also contain lymphokines that suppress the secretion of IgG3 (23) and IgG2 (24,25). These changes in IgG secretion are accompanied by corresponding changes in the steady state levels of mRNA for each IgG subclass (23). BCDF3,-containing SN do not act on cells that bear surface IgG (sIgG) at the initiation of culture (16). Furthermore, BCDF7 does not bind to Sepharose-coupled IgM, IgD, or IgG (25). The cell surface receptor for BCDF3,, then, is probably not sIg.The mechanism of action of BCDF3, is unclear. It might induce growth of an sIgG + subset of B cells. Alternatively, BCDF3" might induce sIgG-B cells to secrete IgG1. In the present studies, we have examined the effects of a BCDFTcontaining T cell SN on normal B cells in a limiting dilution culture system. Our results suggest that BCDF3,-containing SN induce IgG1 secretion in a subset of sIgG-B cells already committed to a differentiation pathway leading to IgG1 secretion. These cells do not arise from precursors of IgG3-secreting cells. Materials and MethodsAnimals. We used female C57BL/6 × DBA/2 F~ (BDF1) mice (The Jackson Laboratory, Bar Harbor, ME) 8-16 wk old. Lewis rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), used at 3-6 wk, were a source of thymus cells.

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“…PFC Assay. We performed reverse PFC assays in 96-well microtiter plates (Linbro Chemical Co., Hamden, CT) by the method of Pike et al (32). We prepared protein A-…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

Layton¹,

Vitetta²,

Uhr³

et al. 1984

The Journal of Experimental Medicine

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“…The cells were pulsed ('H-thymidine; I /tCi/well) 5-6 h before harvesting, and the radiolabel incor porated into 5% trichloroacetic acid-precipitable material was de termined by liquid scintillation counting. The ability of Con ASn to assist B lymphocytes to form plaque-forming cells (PFC) was car ried out according to the methods described by Pike et al (6).…”

Section: Methodsmentioning

confidence: 99%

Immunomodulatory Effects of Orally-Administered Saponins and Nonspecific Resistance against Rabies Infection

Chavali¹,

Campbell²

1987

Int Arch Allergy Immunol

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We present evidence that orally fed Quillaja saponins offer nonspecific resistance to mice against rabies viral infection. Adoptive transfer of spleen cells and thymocytes from animals preconditioned with saponin (SAP), inactivated rabies antigen (AG), or a mixture of AG+SAP has offered significant protection against an intracerebral challenge with live rabies virus. Levels of serum rabies-neutralizing antibodies in the different groups of recipient animals did not correlate with the respective survival rates. Culture supernatants of concanavalin A-stimulated spleen cells from animals fed SAP or AG+SAP induced marked T cell and B cell proliferation, and also greatly enhanced the plaque-forming cell activity of unprimed spleen B cells. Irrespective of the presence or absence of rabies-specific antibodies, sera from animals fed a mixture of AG+SAP induced significant levels of cell proliferation and augmented phytohemagglutinin (PHA)-induced responsiveness of spleen lymphocytes in vitro. The addition of sera from animals fed AG alone resulted in inhibition of cell proliferation and suppressed PH A-induced responses.

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“…Anti-theta-and complement-treated spleen cell suspensions were cultured in limiting numbers in RPMI 1640 supplemented with glutamine, antibiotics, 10% fetal calf serum, and 2 × 10 -5 2-mercaptoethanol in the presence of irradiated rat thymus filter cells at a concentration of 3 × 106 cells and either 50 ~g/ml of LPS or the indicated numbers of 6B7 helper T cells. At each cell dose, 24 or 48 replicate cultures were set up and assayed at day 6 for Ig-secretion plaque-forming cells (PFC) by using a staphylococcal protein A plaque assay (10) according to the method described by Pike et al (11). Alternatively, cultures were continued to days 10-12 and the culture supernatants tested for the presence of M-460 idiotype by a hemagglutination (HA) assay using monoclonal antiidiotype-coated sheep erythrocytes (F6(51)-SRBC) and uncoated SRBC as control.…”

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confidence: 99%

The B cell repertoire revealed by major histocompatibility complex-specific helper T cells. I. Frequencies of a genetically defined V region marker among mitogen- and T helper cell-reactive B lymphocytes in normal and immunized mice.

Primi¹,

Cazenave²

1984

The Journal of Experimental Medicine

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The quantitative description of the V gene repertoire in various steady states is of paramount importance not only for our understanding of the laws that govern the selection of the available repertoire from the pool of germ line encoded V genes, but also for predicting the consequences of specific and nonspecific immunological manipulations.The numbers of cell precursors of clones secreting Ig with a given specificity cannot be directly evaluated in mass cultures or in adoptive transfer experiments, as the number of antibody-producing plaque-forming cells (PFC) i or antibody titer depends on both the number of reacting B cells and on clone sizes. Antibody repertoires can therefore only be estimated under limiting dilution conditions in which lymphocytes are either specifically or polyclonally activated. As it has been generally assumed that B cells sensitive to lipopolysaccharide (LPS) express the total repertoire of V region sets on Ig molecules, this ligand has been considered the mitogen of choice for frequency determinations (1).Recently, however, it has become evident that the repertoire of LPS-sensitive B cells is not completely representative of the total set of Ig specificities in one animal's B cell population and, in addition, it has also been proved that V genes are not, as previously thought, randomly distributed among functional B cell subsets (2, 3). The most important limitation placed on the analysis of the repertoire of LPS-reactive precursor cells, however, is that these lymphocytes may mainly represent newly born short-lived B cells not yet selected into the functional immune system (4-7). This selection may in fact require further differentiation, which enables the cells to become longer living and capable of collaborating with T cells.In order to obtain accurate information on the set of V genes of cells selected

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A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures

Cited by 12 publications

References 16 publications

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

Immunomodulatory Effects of Orally-Administered Saponins and Nonspecific Resistance against Rabies Infection

The B cell repertoire revealed by major histocompatibility complex-specific helper T cells. I. Frequencies of a genetically defined V region marker among mitogen- and T helper cell-reactive B lymphocytes in normal and immunized mice.

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