Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay.
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.
Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba, Japan.
Makoto
M
Arai
Miho
M
Suzuki-Koike
Shin-Ichi
S
Ohtake
Hiroyoshi
H
Ohba
Kunio
K
Tanaka
Joe
J
Chib
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D002413:Cations, Divalent; D003602:Cytotoxicity, Immunologic; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006434:Hemocytes / Q000276:immunology*; D006458:Hemolymph / Q000276:immunology; D007118:Immunoassay / Q000379:methods; D008856:Microscopy, Fluorescence; D012680:Sensitivity and Specificity; D013997:Time Factors; D014561:Urochordata / Q000276:immunology*
D000954:Antigens, Surface; D002413:Cations, Divalent; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM