Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods.
Indirect immunofluorescence techniques for labeling cell surface antigens on murine pluripotent hemopoietic stem cells often result in a reduction of CFU-S numbers. This phenomenon was investigated by comparing indirect immunofluorescence and biotin-avidin methods using anti-T200 and anti-H-2Kk monoclonal antibodies. Mouse bone marrow cells treated with these monoclonal antibodies, alone or in combination with fluorescein conjugates of rabbit antirat or goat antimouse immunoglobulins, respectively, showed reduced numbers of CFU-S. The reduction in CFU-S numbers by anti-H-2Kk antibodies was dependent on the concentration of antibody and on the antigen density on the cells. Near complete CFU-S recovery was obtained with biotin-labeled antibodies and avidin-fluorescein isothiocyanate. The CFU-S recovery obtained was higher with higher numbers of biotin moieties per antibody molecule. Biotinylation itself did not influence the antibody binding properties. The protective effect was independent of the avidin-FITC concentration. Injection of carrageenan, an agent known to block macrophage activity, prevents the reduction of CFU-S recovery caused by anti-H-2Kk antibody treatment. The biotin-avidin procedure permits the measurement of antigen density on pluripotent stem cells through flow cytometry and sorting and full recovery of CFU-S in the in vivo assay.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D001360:Avidin; D001710:Biotin; D003114:Colony-Forming Units Assay; D005069:Evaluation Studies as Topic; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000031:analogs & derivatives*; D013154:Spleen / Q000166:cytology* / Q000276:immunology; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005452:Fluoresceins; D013861:Thiocyanates; D001360:Avidin; D001710:Biotin; D010047:Ovalbumin; D016650:Fluorescein-5-isothiocyanate