Mutual and Directional Allogeneic Cytotoxic Reaction of Hemocytes in the Solitary Ascidian Halocynthia roretzi Revealed by One-step Quantitative Fluorimetric Assay

Abstract

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls alloreactivity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with "effector cells" and those from the other animal of the pair with "target cells". In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.

Study of Color Variation in the Solitary Ascidian Halocynthia roretzi, Collected in the Inland Sea of Japan

“…4, both A4 and A16 lines of wells showed strong colors. As Arai et al (2002) mentioned the correlation between the degree of brown pigmentaion and the intensity of cytotoxicity, there may exist some different cytotoxicity levels among alloreactivity-positive combinations and there may be animals possessing high-cytotoxicity and the ones having lower cytotoxicity. However, we checked only the existence or non-existence of the alloreactivity in this study.…”