Assay to measure CD59 mutations in CHO AL cells using flow cytometry

Background: A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO A(L)) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 1 I but it requires the use of rabbit complement and colony growth for mutant selection. We have developed a more rapid flow cytometry-based mutation assay with CHO AL cells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation. Methods: CHO A(L) cells were treated with gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then allowed to grow for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry. Results: Negative and positive populations were separated by over 100-fold. Mixing various proportions of CD59-positive and -negative cells demonstrated that the assay is highly linear (r(2)=0.9999) and sensitive (<0.05% background mutants). The yield of CD59-inducible mutants was linearly related to dose for a clastogen (gamma-radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific. Conclusions: Mutations induced by genotoxic agents can be rapidly and sensitively measured in CHO A(L) cells using flow cytometry. (C) 2005 Wiley-Liss, Inc.