Enumeration and characterization of virus-specific B cells by multicolor flow cytometry

Virginia P. Doucett, Walter Gerhard, Kristina Owler, Dyan Curry, Lorena Brown, Nicole Baumgarth

Research output: Contribution to journalArticle

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Abstract

To better characterize B cell responses induced to influenza virus, we developed an assay to directly quantify and characterize virus-specific B cells. We used purified and biotinylated whole virus as well as the major influenza virus surface antigen, hemagglutinin (HA) to label virus-specific B cells induced by immunization of mice with whole influenza virus in adjuvant. Immunization with adjuvant alone caused non-specific binding of whole virus to a large number of B cells in the draining lymph nodes as assessed by flow cytometry. This precluded the use of whole virus as a specific staining reagent. In contrast, staining with bromelain-cleaved purified and biotinylated influenza virus HA identified a small population of B cells (roughly 1%) only in the draining lymph nodes of virus-immunized mice. FACS-purification and subsequent ELISPOT analysis showed that HA-labeled B cells contained the vast majority of virus-specific antibody-secreting cells at day 10 after immunization. Overall, virus-specific antibody-secreting cells comprised roughly 10% of the HA-labeled cells. Using HA-staining in conjunction with 8-color flow cytometry we further demonstrated that close to 90% of the HA-labeled cells were CD19+ IgD- CD23- CD24high CD38low germinal center B cells, many of which had incorporated bromodeoxyuridine, indicating recent cell division in vivo. We conclude that viral HA can be used in conjunction with cell surface and intracytoplasmic stains in multicolor flow cytometry to provide detailed phenotypic and functional information on virus HA-specific B cells.

Original languageEnglish (US)
Pages (from-to)40-52
Number of pages13
JournalJournal of Immunological Methods
Volume303
Issue number1-2
DOIs
StatePublished - Aug 2005

Fingerprint

Hemagglutinins
Flow Cytometry
B-Lymphocytes
Viruses
Orthomyxoviridae
Immunization
Antibody-Producing Cells
Staining and Labeling
Viral Hemagglutinins
Lymph Nodes
Bromelains
Enzyme-Linked Immunospot Assay
Virus Attachment
Immunoglobulin D
Germinal Center
Bromodeoxyuridine
Surface Antigens
Cell Division
Coloring Agents
Color

Keywords

  • B cell-specificity
  • Immune response
  • Influenza

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Enumeration and characterization of virus-specific B cells by multicolor flow cytometry. / Doucett, Virginia P.; Gerhard, Walter; Owler, Kristina; Curry, Dyan; Brown, Lorena; Baumgarth, Nicole.

In: Journal of Immunological Methods, Vol. 303, No. 1-2, 08.2005, p. 40-52.

Research output: Contribution to journalArticle

Doucett, Virginia P. ; Gerhard, Walter ; Owler, Kristina ; Curry, Dyan ; Brown, Lorena ; Baumgarth, Nicole. / Enumeration and characterization of virus-specific B cells by multicolor flow cytometry. In: Journal of Immunological Methods. 2005 ; Vol. 303, No. 1-2. pp. 40-52.
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