Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry

Area of Science:

• Genotoxicology
• Mammalian cell mutation assays
• Flow cytometry applications

Background:

• Previous mammalian cell mutation assays using CHO A(L) cells relied on rabbit complement and colony growth.
• These assays measured mutations in the CD59 gene on human chromosome 11.
• The established methods were less rapid and required colony formation for mutant selection.

Purpose of the Study:

• To develop a more rapid and sensitive flow cytometry-based mutation assay using CHO A(L) cells.
• To detect mutations in the CD59 gene without the need for colony formation.
• To utilize monoclonal antibodies against CD59 for mutant detection.

Main Methods:

• CHO A(L) cells were exposed to gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
• Cells were cultured for varying periods to allow for mutant expression.
• Monoclonal antibodies against CD59 were used for cell labeling, followed by flow cytometry analysis.

Main Results:

• Flow cytometry effectively separated CD59-positive and -negative cell populations (over 100-fold).
• The assay demonstrated high linearity (r2 = 0.9999) and sensitivity (<0.05% background mutants).
• Mutant yield correlated linearly with genotoxic agent dose and was specific to treatment and time.

Conclusions:

• Flow cytometry provides a rapid and sensitive method for measuring mutations induced by genotoxic agents in CHO A(L) cells.
• This new assay overcomes limitations of previous methods by eliminating the need for colony growth.
• The technique is suitable for assessing the mutagenic potential of various chemical and physical agents.