Analysis of the limiting-dilution assay used for estimating frequencies of immunocompetent units

Halsall, M.K.; Makinodan, Takashi

doi:10.1016/0008-8749(74)90043-4

Cited by 11 publications

(4 citation statements)

References 19 publications

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“…This was due to a substantial cell loss during the treatment of small numbers of purified spleen cells with collagenase, although collagenase did not affect the viability of the cells (19) . A problem encountered in culturing low cell numbers is the lower than expected PFC response by low numbers of cells to antigenic stimulation as depicted by the shoulder in limiting dilution curves (for review see 28) . In our microculture system, there was also no linear cell dose response relationship .…”

Section: Discussionmentioning

confidence: 99%

Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors.

Haas¹

1975

The Journal of Experimental Medicine

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Clonal selection theories have postulated that individual immunocompetent cells are precommited to the formation of antibodies of a single specificity (1, 2) . There is now experimental evidence that only a distinct subpopulation of lymphocytes can bind and be stimulated by a particular antigen (for review see 3) . Furthermore, it has been shown that individual lymphocytes display only one type of receptor, homogeneous in respect to specificity (4) . This strongly suggests that the precommitment of lymphocytes is expressed by surface receptors for antigen .Depletion of antigen-binding cells by adsorption to antigen-coated beads (5-7) and specific inactivation by radiation (8, 9), provided direct evidence for the specific function of antigen-binding cells in humoral immune responses . However, experiments with lymphocytes depleted in functionally active antigen-specific cells do not prove finally the precommitment hypothesis . Furthermore they do not allow studies on the mechanism of the induction of an immune response . However, isolation and enrichment of lymphocytes bearing surface receptors of a single specificity would be an obvious advantage in studies of the interaction of specific lymphocytes with immunogens, tolerogens, or mitogens . Furthermore, purification of antigen-specific cells would be essential to dissect the various specific and nonspecific interactions of lymphocytes in immune responses .Several attempts have been made to purify antigen-specific lymphocytes and to study their function . Lymphocyte populations from unimmunized mice enriched in sheep red blood cell rosette-forming cells (SRBC-RFC)' contained anti-SRBC antibody-forming cell precursors (AFCP), whose capacity to proliferate and differentiate in vitro (10) or in vivo (11) into antibody-forming cells was dependent on the presence of collaborating cells .Lactoside-specific cells from unimmunized mice were retained in a lactoside-coupled polyacrylamide bead column and could be eluted by an excess of free haptens . The eluted cells were highly enriched in antilactoside AFCP as determined in an adoptive transfer

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Section: Discussionmentioning

confidence: 99%

Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors.

Haas¹

1975

The Journal of Experimental Medicine

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“…In vitro limiting dilution assay was conducted as reported . The glioma cells were serially diluted to the final cell concentrations (10,000, 1000, 100, 10, 1 cells/well in 100 μL of culture medium) in culture medium and added to the 96-well plate on day 0.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro limiting dilution assay was conducted as reported. 36 The glioma cells were serially diluted to the final cell concentrations (10,000, 1000, 100, 10, 1 cells/well in 100 μL of culture medium) in culture medium and added to the 96-well plate on day 0. After one day of incubation, indicated doses of C16 were added to each well and the cells were cultured for another 10 days.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Discovery of Novel N-(Anthracen-9-ylmethyl) Benzamide Derivatives as ZNF207 Inhibitors Promising in Treating Glioma

Zhang,

Ding,

Gao

et al. 2024

J. Med. Chem.

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Targeting tumor stemness is an innovative approach to cancer treatment. Zinc Finger Protein 207 (ZNF207) is a promising target for weakening the stemness of glioma cells. Here, a series of novel N-(anthracen-9-ylmethyl) benzamide derivatives against ZNF207 were rationally designed and synthesized. The inhibitory activity was evaluated, and their structure−activity relationships were summarized. Among them, C16 exhibited the most potent inhibitory activity, as evidenced by its IC 50 values ranging from 0.5−2.5 μM for inhibiting sphere formation and 0.5− 15 μM for cytotoxicity. Furthermore, we found that C16 could hinder tumorigenesis and migration and promote apoptosis in vitro. These effects were attributed to the downregulation of stem-related genes. The in vivo evaluation demonstrated that C16 exhibited efficient permeability across the blood−brain barrier and potent efficacy in both subcutaneous and orthotopic glioma tumor models. Hence, C16 may serve as a potential lead compound targeting ZNF207 and has promising therapeutic potential for glioma.

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“…Estimates of ARC frequency were determined for each responding cell population by the linear regression analysis of the Poisson distribution (Halsall & Makinodan 1974. The probability of a nonresponse is given by the zero order term, Po = e-vN, where v=frequency of ARC and N = t h e number of responding lymphocytes per well.…”

Section: Methodsmentioning

confidence: 99%

Limiting Dilution Analysis of Alloantigen‐Reactive Cells which Respond to Allogeneic Lymphocytes in Human MLC and PLT

1980

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We have determined the frequency of the alloantigen-reactive cells (ARC) in human ILC and PLT by the limiting dilution analysis. In PLT, the frequency of the ARC to the original sensitizing donor ranged between 1:32 to 1:62, an increase of six to nine-fold after priming in MLC. The MLC primed populations were also enriched (three to five fold) for the ARC responding to the PL-positive allogeneic donors. The incidence of the ARC was 1 6 2 to 1:118 with donors positive for the sensitizing HLA-DRw antigen and 1:77 to 1:144 with donors negative for the specific HLA-DRw determinant. The results from experiments utilizing pooled stimulating cells from the original and allogeneic donors suggest that same subpopulation of cells responds to :he sensitizing HLA-DRw determinant, whether it is presented by the specific stimulator or by a thirdparty allogeneic donor. On the other hand, different subpopulations of alIoreactive cells respond t o different alloantigens.In MLC experiments between HLA-identical siblings, the incidence of the ARC ranged between 1:995 to 1:1673. The responses of the ARC to non-HLA antigens were observed under conditions where responder cells were limiting. Also, the responses of the limiting numbers of responding cells were inhibited by mitomycin-treated autologous lymphocytes. Nonresponse in MLC combinations with higher responder cell numbers was not due to small numbers of stimulating cells.

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Analysis of the limiting-dilution assay used for estimating frequencies of immunocompetent units

Cited by 11 publications

References 19 publications

Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors.

Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors.

Discovery of Novel N-(Anthracen-9-ylmethyl) Benzamide Derivatives as ZNF207 Inhibitors Promising in Treating Glioma

Limiting Dilution Analysis of Alloantigen‐Reactive Cells which Respond to Allogeneic Lymphocytes in Human MLC and PLT

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