A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62.
Bone marrow (BM)-derived dendritic cells (DC) are the most potent known antigen (Ag) presenting cell in vivo and in vitro. Detailed analysis of their properties and mechanisms of action requires an ability to produce large numbers of DC. Although DC have been isolated from several rat tissues, including BM, the yield is uniformly low. We describe a simple method for the propagation of large numbers of DC from rat BM and document cell yield with the rat DC marker, OX-62. After depletion of plastic-adherent and Fc+ cells by panning on dishes coated with normal serum, residual BM cells were cultured in gelatin coated flasks using murine rGM-CSF supplemented medium. Prior to analysis, non-adherent cells were re-depleted of contaminating Fc+ cells. Propagation of DC was monitored by double staining for FACS analysis (major histocompatibility complex (MHC) class II+/OX-62+, OX-19-). Functional assay, morphological analysis and evaluation of homing patterns of cultured cells revealed typical DC characteristics. MHC class II and OX-62 antigen expression increased with time in culture and correlated with allostimulatory ability. DC yield increased until day 7, when 3.3 x 10(6) DC were obtained from an initial 3 x 10(8) unfractionated BM cells. Significant numbers of DC can be generated from rat BM using these simple methods. This should permit analysis and manipulation of rat DC functions in vivo and in vitro.
Pittsburgh Transplantation Institute, University of Pittsburgh Health Science Center, PA 15261.
M
M
Chen-Woan
C P
CP
Delaney
V
V
Fournier
Y
Y
Wakizaka
N
N
Murase
J
J
Fung
T E
TE
Starzl
A J
AJ
Demetris
D000818:Animals; D000911:Antibodies, Monoclonal; D001854:Bone Marrow Cells; D002448:Cell Adhesion; D002455:Cell Division / Q000187:drug effects; D046508:Culture Techniques / Q000379:methods; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology* / Q000648:ultrastructure; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005780:Gelatin / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D007959:Lymphocyte Culture Test, Mixed; D010969:Plastics; D051381:Rats; D011917:Rats, Inbred Lew; D011961:Receptors, Fc / Q000276:immunology
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D010969:Plastics; D011961:Receptors, Fc; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005780:Gelatin