Mixed-Incubation of Allogeneic Hemocytes in Tunicate Halocynthia roretzi

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We developed a new convenient method to examine self-nonself compatibility of tunicate, Halocynthia roretzi, by reactions in a mixture of hemocytes. Tunicate hemolymph samples of 100μl each, harvested from two individuals and containing approximately 10^6 cells, were mixed and incubated for 12-20hr at 15-23℃. This incubation of hemocyte-mixture resulted in two definite types of phenomena : i. e. , presence and absence of alloreactivity which was represented by intensive discharge of hemocyte contents and amorphous precipitation of hemocyte clusters with brown pigmentation. Such alloreactivity exsisted in most nonself combinations, while the incubation of hemocytes from single tunicate (autologous control) did not exhibit any of them. Autologous controls and certain several nonself combinations showed a simple accumulation of hemocytes, as forming buttons at the center of U-bottomed culture wells. Consequently, those nonself combinations were judged as non-reactive or compatible. Autologous controls and the non-reactive nonself combinations formed large spherical aggregates which did not adhere to the culture wells and were evidently different from the amorphous clusters found in alloreactive hemocyte mixtures. Furthermore, we confirmed the consistency between the alloreactivity measured by mixed-hemocyte-incubation (MHI-assay) and that measured by contact reaction. The procedure for MHI-assay in U-bottomed culture plate was simple, and compatibility could be judged without microscopic observation. Thus, MHI-assay will be an effective method to test self-nonself compatibility of numerous number of combinations using a large number of tunicates, that will be required in studying genetic background of tunicate allogeneic recognition.
社団法人日本動物学会の論文
1994-12-15

Mixed-Incubation of Allogeneic Hemocytes in Tunicate Halocynthia roretzi

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