Isolation of functionally active murine follicular dendritic cells.
Biochemical, genetic, and immunological studies of follicular dendritic cells (FDCs) have been hampered by difficulty in obtaining adequate numbers of purified cells in a functional state. To address this obstacle, we enriched FDCs by irradiating mice to destroy most lymphocytes, excised the lymph nodes, and gently digested the nodes with an enzyme cocktail to form single cell suspensions. The FDCs in suspension were selected using the specific mAb FDC-M1 with magnetic cell separation technology. We were able to get nearly a million viable lymph node FDCs per mouse at about 90% purity. When examined under light and transmission electron microscopy, the cytological features were characteristic of FDCs. Furthermore, the cells were able to trap and retain immune complexes and were positive for important phenotypic markers including FDC-M1, CD21/35, CD32, CD40, and CD54. Moreover, the purified FDCs exhibited classical FDC accessory activities including: the ability to co-stimulate B cell proliferation, augment antibody responses induced by mitogens or antigens, maintain B cell viability for weeks, and protect B lymphocytes from anti-FAS induced apoptosis. In short, this combination of methods made it possible to obtain a substantial number of highly enriched functional murine FDCs.
Sukumar
Selvakumar
S
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, 23298-0678, USA.
Szakal
Andras K
AK
Tew
John G
JG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis; D000954:Antigens, Surface / Q000276:immunology; D017209:Apoptosis / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D002452:Cell Count; D002467:Cell Nucleus / Q000648:ultrastructure; D049109:Cell Proliferation; D022081:Cell Surface Extensions / Q000648:ultrastructure; D002470:Cell Survival; D018920:Coculture Techniques; D020566:Dendritic Cells, Follicular / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D007074:Immunoglobulin G / Q000276:immunology; D018189:Immunomagnetic Separation / Q000379:methods*; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D010047:Ovalbumin / Q000276:immunology; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000954:Antigens, Surface; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D010047:Ovalbumin