Further improvement of dye-exclusion microcytotoxicity assay by introducing low melting point agarose into the medium

We developed an improved method for the dye-exclusion microcytotoxicity test by introducing low melting point agarose (LMP-Aga) into the assay medium. The procedures in our method are made very feasible by such noble characteristics of LMP-Aga that once solubilized, the sol state is retained at 37 degrees C, while once gelated, the gelated state is retained even at 37 degrees C. The most important advantage of our method is that even distribution, without any accumulation, of the target cells on the bottom surface of the test wells and no dislodgement of the target cells from the wells by mechanical force inflicted during the assay, enable us to observe the target cells precisely and to obtain an accurate percentage of dead target cells. Another advantage of the present method is that in complement-mediated cytotoxicity tests, it is possible to use unabsorbed normal serum as complement source because naturally occurring cytotoxicity in normal serum is completely removed during the diffusion into the LMP-Aga medium. Thus it should be concluded that our method is excellent in all respects, e.g., accuracy and reproducibility in the results of the test, and feasibility of the procedures.