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A System for Quantitative Determination of Cytotoxic Activity of Antisera to Ascites Tumor Cells^*

( Biochemistry Section, Department of Surgery, Tufts University School of Medicine, Boston City Hospital; and the Mallory Institute of Pathology, Boston City Hospital, Boston, Mass.)

Criteria are described for recognizing early degenerative changes that occur when mouse ascites tumor cells are exposed to homologous or heterologous antisera. These permit determination of the same percentage of deformed cells, irrespective of whether or not vital staining dyes are used.

When 10 million cells of the L4946 or E9514-A ascites tumors were incubated at 37° C. in 1 ml. modified Locke's buffer with excess complement, the percentage of deformed cells leveled out between 1 and 3 hours' incubation at a value determined by antibody concentration. A titration curve, of percentage cells deformed after 2 hours' incubation against antiserum concentration, gave the cytotoxic titer, i.e., the final concentration of antiserum giving 50 per cent plus one-half the control level of deformed cells. For a rabbit antiserum to L4946 cells, the cytotoxic titer was 1.24 parts per thousand, with a standard deviation of 21 per cent. A relationship between cytotoxic titer and cell concentration in the medium was demonstrated, and dependence on pH, osmolarity, and complement concentration were investigated.

To relate cell deformation to cell death, serial dilutions of L4946 cells containing known percentages of deformed cells were inoculated into AKR mice and survival noted. The results indicate that the microscopic count underestimates the damage done to cells by antiserum. A method for estimating cell viability following inoculation of mice with tumor cells is described, which takes into account the period of temporary survival of mice dying from tumor, in addition to mere death or long-term survival.

^* This investigation was supported by the National Cancer Institute of the National Institutes of Health, United States Public Health Service, through grant no. CY-4469(C1) A&I.

Received 3/30/60.