A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation.
The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Cells from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10%, representing an approximately 15-fold enrichment compared to unseparated spleen cells (< 1% DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33D1-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33D1-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the MHC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.
Ossevoort
M A
MA
Department of Immunohematology and Bloodbank, University Hospital, Leiden, Netherlands.
Toes
R E
RE
De Bruijn
M L
ML
Melief
C J
CJ
Figdor
C G
CG
Kast
W M
WM
D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D010222:Parainfluenza Virus 1, Human / Q000276:immunology; D013154:Spleen / Q000166:cytology* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology