Determination of mixed chimerism by a simple flow cytometry method.
A simple, sensitive and accurate method was developed to determine the level of lymphoid chimerism in bone marrow-transplanted rodents. The method is based on flow cytometry using polyclonal alloantisera and labeled second step anti-IgG antibodies. Using mixtures of spleen cells from different mouse strains, it was demonstrated that low levels of chimeric cells (less than 1%) could easily be detected. Moreover, using two-color fluorescence analysis, the level of chimerism could also be determined in subpopulations of lymphoid cells, e.g., CD4 or CD8 cells and was found to be identical to the results obtained in unseparated lymphoid populations. The method was compared to the complement dependent cytotoxicity assay (CDCA) and to the flow cytometric determination of chimerism using labeled monoclonal antibodies against specific MHC antigens. CDCA was found to be more labor intensive and could only estimate the composition of the cell mixtures without detecting low levels of chimerism (less than 5%). The results of flow cytometry, using directly labeled monoclonal antibodies or polyclonal antibodies with second step reagents, were identical. It is concluded that, due to its simplicity and high sensitivity, the method described permits reliable determination of the level of mixed chimerism in rodents and is an excellent alternative when no anti-MHC mAbs are available.
Leenarets
P L
PL
Division of Nephrology, University of Leuven, Belgium.
Vandeputte
M
M
Waer
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016026:Bone Marrow Transplantation / Q000276:immunology; D002678:Chimera / Q000276:immunology*; D003165:Complement System Proteins / Q000502:physiology; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods; D006649:Histocompatibility Antigens / Q000032:analysis; D007106:Immune Sera; D007074:Immunoglobulin G / Q000032:analysis; D007158:Immunologic Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL
D000911:Antibodies, Monoclonal; D006649:Histocompatibility Antigens; D007106:Immune Sera; D007074:Immunoglobulin G; D003165:Complement System Proteins