Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen.
Dendritic cells are a specialized but trace population of antigen presenting cells that always have been enriched by multi-step procedures over a period of 1 or more days in tissue culture. Here we describe the isolation of dendritic cells from fresh mouse spleen suspensions using the FACS and a monoclonal antibody, N418, to the p150/90 member of the leukocyte integrin family (Metlay et al., 1990). By two color fluorescence activated cell sorter (FACS) analyses, the trace N418+ subset expressed most of the surface markers, including the 33D1 antigen, that are characteristic of dendritic cells isolated by other methods. An exception was that small amounts of Fc receptors, CD4 and F4/80 antigen were detected initially, but these diminished upon culture. In functional assays, sorted N418+ cells from fresh spleen were at least 30 times more active than N418- cells in presenting antigen to T cells. The assays were stimulation of the primary mixed leukocyte reaction and presentation of exogenous protein antigens to sensitized populations of lymph node T cells. The viability and MLR stimulating function of the sorted populations both were increased upon exposure to the cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). These results indicate that dendritic cells can be enriched from fresh isolates of mouse spleen using the FACS, and that when this is done, many of the distinctive features of dendritic cells - phenotype, APC function, and sensitivity to appropriate cytokines - are apparent.
Crowley
M T
MT
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Inaba
K
K
Witmer-Pack
M D
MD
Gezelter
S
S
Steinman
R M
RM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016023:Integrins / Q000276:immunology; D007515:Islets of Langerhans / Q000166:cytology; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010641:Phenotype; D013154:Spleen / Q000166:cytology*
D000911:Antibodies, Monoclonal; D016207:Cytokines; D016023:Integrins