TY - JOUR
T1 - A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate
AU - Raveh, Dina
AU - Huberman, Eliezer
N1 - Funding Information:
We wish to thank S. Macguire and especially Drs. A. Lampert and B.A. Carnes for help with statistics, R. Feldman for useful discussions, and G. Raziel for photographic reproductions. This work was supported by grants from the Israel Academy of Science, the P. Sapir Fund of Mifal Hapayis, by funds provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health (U.S.), under contract No. 1-CO-65341 (International Cancer Research Technology Transfer, ICRETT) to D. Raveh, and by the Office of Health and Environmental Research, U.S. Department of Energy, under contract No. W-3 l-109-ENG-38 and by the National Cancer Institute contract No. Y01-CP. 70222 to E. Huberman.
PY - 1983/1/1
Y1 - 1983/1/1
N2 - 6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (103 - 104 cells/0.2 ml growth medium/0.4 cm2) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 105 colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 105 colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.
AB - 6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (103 - 104 cells/0.2 ml growth medium/0.4 cm2) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 105 colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 105 colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.
UR - http://www.scopus.com/inward/record.url?scp=0020638312&partnerID=8YFLogxK
U2 - 10.1016/0165-1161(83)90004-3
DO - 10.1016/0165-1161(83)90004-3
M3 - Article
AN - SCOPUS:0020638312
SN - 0165-1161
VL - 113
SP - 499
EP - 506
JO - Mutation Research - Environmental Mutagenesis and Related Subjects Including Methodology
JF - Mutation Research - Environmental Mutagenesis and Related Subjects Including Methodology
IS - 6
ER -