A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.
A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.
D000818:Animals; D000941:Antigens; D001665:Binding Sites; D002469:Cell Separation; D002498:Centrifugation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006462:Hemolytic Plaque Technique; D051379:Mice; D008808:Mice, Inbred CBA; D012397:Rosette Formation; D012756:Sheep; D013154:Spleen / Q000276:immunology; D013997:Time Factors
D000941:Antigens