Immune cytolysis in relation to the growth cycle of Chinese hamster cells.

provided by Dr. M. M. Elkind. The colony-forming assay techniques were essentially those of Elkind (6, 7). These cells grow attached to glass or plastic, have a fibroblast morphology, double in about 8 hr, and produce, with high plating efficiency, colonies about 3 mm in diameter in 7 days. Cells were plated in 9-cm Petri dishes (Falcon Plastic #3003, Falcon Plastics, Oxnard, Calif.,) using EM-l5, a modified Eagle's medium (3), plus 15% fetal calf serum (Flow Laboratories, Rockville, Md.). To help ensure that the distribution of cells throughout their growth cycle was close to that resulting from asynchronous, log phase growth, they were incubated overnight before each experiment was started. Since the overnight growth period increased the number of cells about 4-fold, the experiments were performed on populations of microcolonies rather than on single cells. Following treatment, cells were incubated for 7 days, an interval giving a maximum yield of colonies. The colonies were stained with methylene blue and counted. Tests for contamination of the cell line with Mycoplasma were negative. Cell Synchrony and Radiation. For evaluation of the influence of the position in the cell cycle on susceptibility to immune cytolysis, target cells were pretreated with 2 mM hydroxyurea for 3.5 hr. As has been shown in detail by both Elkind et a!. (4, 5) and Sinclair (16) with this cell line, this treatment synchronizes the potential colony-forming cells at the G1 -S border. Following removal of hydroxyurea, the cells progress synchronously through their cell cycle. After 3 hr, they are in the middle of the S phase, and in another 3.5 to 5 hr they are in G2 mitosis (5) Cells were irradiated with 55-ky X-rays at a dose rate of 722 rads/min filtered by the 1-mm Be exit window of the tube plus 0.175 mm Al as described by Elkind (6). To minimize absorption effects of this irradiation by condensed matter, cells were irradiated after removal of the overlying medium. Immunological Techniques. Hyperimmune serum prepared against whole CHL cells in a New Zealand rabbit was kindly provided by Dr. A. R. Baker and Dr. H. R. Colten (2). The rabbit anti-CHL cell antiserum was heat-inactivated at 56°for 30 mm and diluted in EM-lS. CHL cells, either in asynchronous exponential growth or at various times after hydroxyurea synchronization, were sensitized by exposure to antiserum for 60 or 180 mm at 37° with varying concentrations of antiserum in a 10-mi volume. The medium