A flow cytometric technique for quantitation of B-cell activation

Garner, Joseph G.; Colvin, Robert B.; Schooley, Robert T.

doi:10.1016/0022-1759(84)90083-8

Cited by 5 publications

(1 citation statement)

References 12 publications

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“…In recent years several studies have shown that intracellular antigens can be readily investigated by flow cytometry. Highly specific staining has been achieved for cytoskeletal structures such as intermediate filaments (171, for intranuclear proteins such as simian virus 40 T antigen (7) and cytoplasmic immunoglobulin (5,17,221. Situations may arise when it would be advantageous to be able t o simultaneously examine both surface and cytoplasmic cell antigens.…”

Section: Key Terms: Intracytoplasmic and Surface Immunofluorescence mentioning

confidence: 99%

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

Hayden¹,

Miller²,

Wotherspoon³

et al. 1988

Cytometry

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A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates.Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA.STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a crossreactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxidsuppressor T cells and Leu 3a+ helpedinducer T cells but is not induced in the Leu 15" population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells. Key terms: Intracytoplasmic and surface immunofluorescence, flow cytometry analysisIn recent years several studies have shown that intracellular antigens can be readily investigated by flow cytometry. Highly specific staining has been achieved for cytoskeletal structures such as intermediate filaments (171, for intranuclear proteins such as simian virus 40 T antigen (7) and cytoplasmic immunoglobulin (5, 17,221. Situations may arise when it would be advantageous to be able t o simultaneously examine both surface and cytoplasmic cell antigens. One example would be to establish the relationship between a cytoplasmic antigen and the subset derivation of a given lymphocyte population which is largely defined by the surface expression of characteristic epitopes (8). Previous approaches to determine the existence of such relationships have involved the separation of cell subpopulations by either panning on antibody-coated petri dishes (21) or flow cytometric sorting before analysis for cytoplasmic antigen expression. However, the length of time required and the physical nature of such cellular separation techniques may result in a decrease in the number of viable cells and cannot guarantee discrete subset separations. For these reasons a two-colour immunofluorescence staining method was developed to allow the simultaneous analysis of both surface and cytoplasmic cellular antigens.Previously, we reported the detection of a unique cytoplasmic T cell activation protein, Sezary T cell antigen (STA), using flow cytometry (20). STA is a high molecular weight protein (> 200 kilodaltons unreduced) which is detected with a monoclonal antibody, K-1-21. Interestingly, K-1-21 also reacts with a structurally distinct conformational epitope expressed in the constant domain of free, but not heavy chain-associated, human kappa light chains (15) by negative selection panning (21) before cytometric analysis for surface antigens or for...

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Section: Key Terms: Intracytoplasmic and Surface Immunofluorescence mentioning

confidence: 99%

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

Hayden¹,

Miller²,

Wotherspoon³

et al. 1988

Cytometry

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Analysis of Intracellular Antigens by Flow Cytometry: Methods for Cell Permeabilization to Antibodies

Giloh

1993

Flow Cytometry

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Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies.

Bardales¹,

Al‐Katib²,

Carrato³

et al. 1989

J Histochem Cytochem.

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The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.

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A flow cytometric technique for quantitation of B-cell activation

Cited by 5 publications

References 12 publications

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

Analysis of Intracellular Antigens by Flow Cytometry: Methods for Cell Permeabilization to Antibodies

Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies.

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