[Isolation of FDC-lymphocyte clusters from human tonsillar tissues].
Follicular B cells and follicular dendritic cells (FDCs) form FDC-lymphocyte clusters and play a central role in events related to humoral immunity in the lymphoid follicle (LF). The secondary LF is divided into five zones, each of which exhibits functional differences. However, the distribution of the clusters across the five follicular zones remains unclear. We here report a procedure for isolating FDC-lymphocyte clusters from fixed tonsillar tissues and compare of the structure of clusters isolated from three follicular zones: the mantle, light and dark zones. First, the germinal centers (GCs) of the secondary LFs were removed under a stereoscope, and the GCs were enzymatically digested for 20, 30, 40 and 50 minutes at 37 degrees C. The FDC-lymphocyte clusters were then isolated using a discontinuous density gradient and a Magnetic Particle Concentrator, followed by microbeads. The number of isolated medium sized clusters composed of 6-25 cells was greatest when the samples were incubated for 40 minutes. To detect the mantle, light and dark zones, and GCs, isolated FDC-lymphocyte clusters from each zone were immunostained. Their cell structures were then compared. The clusters were composed mainly of B cells (comprising about 80% of the cells in each cluster, on average), T cells, natural killer/T cells and macrophages were also observed, but less frequently. The proportions of CD 45 RO-positive cells and CD4-positive cells were clearly different for each zone, with CD4-positive cells in the majority. No clear differences in isolated clusters from fixed and unfixed tonsillar tissues were observed. Our data indicate that this procedure is suitable for isolating FDC-lymphocyte clusters from fixed lymphoid tissues and that the proportions of cells composing the clusters differ in the three follicular zones.
Takemoto
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Department of Otolaryngology, Tokyo Women's Medical University, Tokyo.
D000917:Antibody Formation; D001402:B-Lymphocytes / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002648:Child; D020566:Dendritic Cells, Follicular / Q000276:immunology; D006801:Humans; D014066:Palatine Tonsil / Q000166:cytology* / Q000276:immunology