title: Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells.
abstract: In multiparameter fluorescence-activated cell sorter studies presented here, we use the highly fluorescent protein phycoerythrin (PE) as an immunizing antigen and as a fluorescent probe to reveal and isolate antigen-binding memory B cells. We demonstrate directly that memory B cells in spleens from PE-primed mice stain brightly with PE and produce strong IgG1 anti-PE responses when sorted and cultured with L3T4+ helper T cells. That is, as few as 500 sorted PE-binding cells per culture well are sufficient to produce high-level anti-PE responses, presenting antigen in a form that induces helper T cells. Three-color immunofluorescence studies reveal that cells staining brightly with PE reside primarily in a population of B220+(IgM,IgD)- cells and comprise 0.02-0.05% of the spleen cells. Complementary studies with 2,4-dinitrophenyl memory B cells show that whereas this B220+-(IgM,IgD)- population (which comprises only 1-2% of the spleen cells) gives high-affinity anti-dinitrophenyl responses, the predominant B220+(IgM,IgD)+ population yields only low-affinity antibody. Finally, we find that although both populations have similar Ia levels, cells in the high-affinity fraction have greatly reduced levels of surface immunoglobulin.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000145:classification / Q000166:cytology / Q000276:immunology*; D002469:Cell Separation; D005434:Flow Cytometry; D007156:Immunologic Memory; D051379:Mice; D008807:Mice, Inbred BALB C; D010799:Phycoerythrin; D010860:Pigments, Biological; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D013154:Spleen / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D010860:Pigments, Biological; D011947:Receptors, Antigen, B-Cell; D010799:Phycoerythrin