Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry

J. M. Cory, B. M. Ohlsson-Wilhelm, E. J. Brock, N. A. Sheaffer, M. E. Steck, M. Elaine Eyster, F. Rapp

    Research output: Contribution to journalArticle

    31 Citations (Scopus)

    Abstract

    Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10-4 following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab′)2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.

    Original languageEnglish (US)
    Pages (from-to)71-78
    Number of pages8
    JournalJournal of Immunological Methods
    Volume105
    Issue number1
    DOIs
    StatePublished - Dec 4 1987

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    Flow Cytometry
    HIV
    Lymphocytes
    Viral Core Proteins
    RNA-Directed DNA Polymerase
    Methanol
    Human Immunodeficiency Virus Proteins
    Fixatives
    Octoxynol
    Indirect Fluorescent Antibody Technique
    Acetone
    Fluorescein
    Goats
    Virion
    Immunoglobulins
    Monoclonal Antibodies
    Antigens
    Cell Line
    Antibodies
    Infection

    All Science Journal Classification (ASJC) codes

    • Immunology and Allergy
    • Immunology

    Cite this

    Cory, J. M. ; Ohlsson-Wilhelm, B. M. ; Brock, E. J. ; Sheaffer, N. A. ; Steck, M. E. ; Eyster, M. Elaine ; Rapp, F. / Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry. In: Journal of Immunological Methods. 1987 ; Vol. 105, No. 1. pp. 71-78.
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    abstract = "Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10-4 following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab′)2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.",
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    Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry. / Cory, J. M.; Ohlsson-Wilhelm, B. M.; Brock, E. J.; Sheaffer, N. A.; Steck, M. E.; Eyster, M. Elaine; Rapp, F.

    In: Journal of Immunological Methods, Vol. 105, No. 1, 04.12.1987, p. 71-78.

    Research output: Contribution to journalArticle

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    AU - Cory, J. M.

    AU - Ohlsson-Wilhelm, B. M.

    AU - Brock, E. J.

    AU - Sheaffer, N. A.

    AU - Steck, M. E.

    AU - Eyster, M. Elaine

    AU - Rapp, F.

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    N2 - Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10-4 following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab′)2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.

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