Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS).
A method is described for the isolation of antigen-specific B cells from immunized and subsequently boosted mice. Antigen-specific B cells were stained by incubation with fluorescein isothiocyanate (FITC)-labelled antigen and then detected and isolated in a fluorescence activated cell sorter (FACS). Ovalbumin (OVA) and Helix pomatia haemocyanin (HPH) were used as antigens in this procedure, yielding relative amounts of antigen-FITC-binding lymphocytes of 0.9 +/- 0.4% and 3.5 +/- 3.1%. The FITC-positive cells were visible as distinct cell populations in the FACS-generated histograms. All antigen-FITC-binding cells were B cells, as shown by double staining with phycoerythrin-conjugated anti-mouse Ig In addition, as tested in a spot-ELISA, the sorted, antigen-FITC-binding cell population contained almost the entire population of antigen-specific antibody-producing B cells. However, sorting had a negative influence on the antibody production capability of the sorted cells. Through washing of isolated spleen cells in the procedure before labelling with antigen-FITC proved to be essential for the specific detection of antigen-specific B cells, since staining without prior washing resulted in antigen-FITC binding to all B cells. This 'nonspecific' staining phenomenon was caused by the presence of antibodies, specific for the immunizing/boosting antigen, which were also present in the spleen cell suspension. These antibodies formed immune complexes with antigen-FITC and bound to Fc receptors present on all B cells, interfering in this way with any specific binding of antigen-FITC to sIg on the B cells.
Hoven
M Y
MY
Department of Clinical Immunology, University Hospital, Groningen, The Netherlands.
De Leij
L
L
Keij
J F
JF
The
T H
TH
D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007114:Immunization; D037102:Lectins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010047:Ovalbumin / Q000276:immunology
D000936:Antigen-Antibody Complex; C038095:Helix lectin; D037102:Lectins; D010047:Ovalbumin