Direct depletion and purification of monoclonal antibody defined cells from unfractionated human mononuclear leukocytes using antibody coated polystyrene Petri dishes

K. D. Cooper, K. Kang, T. L. Bush, Jon Hanifin

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Abstract

Human peripheral blood mononuclear leukocytes were depleted or enriched in various monoclonal antibody-defined subsets using a simplification of the indirect 'panning' technique. Unfractionated mononuclear leukocytes (MNL) were sensitized with appropriate dilutions of monoclonal antibodies to human Lyt3, OKT4 and OKT8 antigens, and to a monocyte-myeloid line antigen. The sensitized cells were then placed on polystyrene Petri dishes coated with goat antimouse IgG to obtain a population of cells depleted and a population of cells enriched in each cell type. Direct separation of surface immunoglobulin-bearing cells was similarly achieved by coating the Petri dish with goat antihuman immunoglobulins. Results showed that MNL could be depleted to greater than 95% purity with these methods and that positively selected adherent cells could be enriched to at least 85% purity. The relative proportions of MNL subpopulations in various sorted cell populations is reported. Cells obtained by panning are functionally intact. As compared to complement lysis both marker positive and marker negative cells can be obtained and the technique requires less technical expertise than using fluorescence activated cell sorting. The modification reported here is simpler and less time consuming for human T cell subpopulation separations than previously reported panning methods since an initial sheep erythrocyte rosetting step was not used.

Original languageEnglish (US)
Pages (from-to)97-100
Number of pages4
JournalJournal of Clinical and Laboratory Immunology
Volume13
Issue number2
StatePublished - 1984

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Mononuclear Leukocytes
Polystyrenes
Monoclonal Antibodies
Antibodies
Goats
Population
Professional Competence
Antigens
B-Cell Antigen Receptors
Cell Separation
Immunoglobulins
Monocytes
Sheep
Flow Cytometry
Immunoglobulin G
Erythrocytes
T-Lymphocytes

ASJC Scopus subject areas

  • Immunology

Cite this

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title = "Direct depletion and purification of monoclonal antibody defined cells from unfractionated human mononuclear leukocytes using antibody coated polystyrene Petri dishes",
abstract = "Human peripheral blood mononuclear leukocytes were depleted or enriched in various monoclonal antibody-defined subsets using a simplification of the indirect 'panning' technique. Unfractionated mononuclear leukocytes (MNL) were sensitized with appropriate dilutions of monoclonal antibodies to human Lyt3, OKT4 and OKT8 antigens, and to a monocyte-myeloid line antigen. The sensitized cells were then placed on polystyrene Petri dishes coated with goat antimouse IgG to obtain a population of cells depleted and a population of cells enriched in each cell type. Direct separation of surface immunoglobulin-bearing cells was similarly achieved by coating the Petri dish with goat antihuman immunoglobulins. Results showed that MNL could be depleted to greater than 95{\%} purity with these methods and that positively selected adherent cells could be enriched to at least 85{\%} purity. The relative proportions of MNL subpopulations in various sorted cell populations is reported. Cells obtained by panning are functionally intact. As compared to complement lysis both marker positive and marker negative cells can be obtained and the technique requires less technical expertise than using fluorescence activated cell sorting. The modification reported here is simpler and less time consuming for human T cell subpopulation separations than previously reported panning methods since an initial sheep erythrocyte rosetting step was not used.",
author = "Cooper, {K. D.} and K. Kang and Bush, {T. L.} and Jon Hanifin",
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T1 - Direct depletion and purification of monoclonal antibody defined cells from unfractionated human mononuclear leukocytes using antibody coated polystyrene Petri dishes

AU - Cooper, K. D.

AU - Kang, K.

AU - Bush, T. L.

AU - Hanifin, Jon

PY - 1984

Y1 - 1984

N2 - Human peripheral blood mononuclear leukocytes were depleted or enriched in various monoclonal antibody-defined subsets using a simplification of the indirect 'panning' technique. Unfractionated mononuclear leukocytes (MNL) were sensitized with appropriate dilutions of monoclonal antibodies to human Lyt3, OKT4 and OKT8 antigens, and to a monocyte-myeloid line antigen. The sensitized cells were then placed on polystyrene Petri dishes coated with goat antimouse IgG to obtain a population of cells depleted and a population of cells enriched in each cell type. Direct separation of surface immunoglobulin-bearing cells was similarly achieved by coating the Petri dish with goat antihuman immunoglobulins. Results showed that MNL could be depleted to greater than 95% purity with these methods and that positively selected adherent cells could be enriched to at least 85% purity. The relative proportions of MNL subpopulations in various sorted cell populations is reported. Cells obtained by panning are functionally intact. As compared to complement lysis both marker positive and marker negative cells can be obtained and the technique requires less technical expertise than using fluorescence activated cell sorting. The modification reported here is simpler and less time consuming for human T cell subpopulation separations than previously reported panning methods since an initial sheep erythrocyte rosetting step was not used.

AB - Human peripheral blood mononuclear leukocytes were depleted or enriched in various monoclonal antibody-defined subsets using a simplification of the indirect 'panning' technique. Unfractionated mononuclear leukocytes (MNL) were sensitized with appropriate dilutions of monoclonal antibodies to human Lyt3, OKT4 and OKT8 antigens, and to a monocyte-myeloid line antigen. The sensitized cells were then placed on polystyrene Petri dishes coated with goat antimouse IgG to obtain a population of cells depleted and a population of cells enriched in each cell type. Direct separation of surface immunoglobulin-bearing cells was similarly achieved by coating the Petri dish with goat antihuman immunoglobulins. Results showed that MNL could be depleted to greater than 95% purity with these methods and that positively selected adherent cells could be enriched to at least 85% purity. The relative proportions of MNL subpopulations in various sorted cell populations is reported. Cells obtained by panning are functionally intact. As compared to complement lysis both marker positive and marker negative cells can be obtained and the technique requires less technical expertise than using fluorescence activated cell sorting. The modification reported here is simpler and less time consuming for human T cell subpopulation separations than previously reported panning methods since an initial sheep erythrocyte rosetting step was not used.

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