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 language | English (US) |
|---|---|
| Pages (from-to) | 40-52 |
| Number of pages | 13 |
| Journal | Journal of Immunological Methods |
| Volume | 303 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Aug 2005 |
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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 journal › Article
}
TY - JOUR
T1 - Enumeration and characterization of virus-specific B cells by multicolor flow cytometry
AU - Doucett, Virginia P.
AU - Gerhard, Walter
AU - Owler, Kristina
AU - Curry, Dyan
AU - Brown, Lorena
AU - Baumgarth, Nicole
PY - 2005/8
Y1 - 2005/8
N2 - 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.
AB - 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.
KW - B cell-specificity
KW - Immune response
KW - Influenza
UR - http://www.scopus.com/inward/record.url?scp=23844456374&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=23844456374&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2005.05.014
DO - 10.1016/j.jim.2005.05.014
M3 - Article
C2 - 16045923
AN - SCOPUS:23844456374
VL - 303
SP - 40
EP - 52
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -