Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.
Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.
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使用流式细胞术测量 CHO A(L) 细胞中的 CD59 突变。
DOI：
10.1002/cyto.a.20168
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
Fox,MichaelH
中科院分区：
文献类型：
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作者：
Ross,CarleyD;Lim,Chang-Uk;Fox,MichaelH
背景利用稳定整合人11号染色体的中国仓鼠卵巢细胞系（CHO AL）建立了一种灵敏的哺乳动物细胞突变检测方法。该检测方法测量11号染色体上CD 59基因的突变，但需要使用兔补体和菌落生长进行突变体选择。我们已经开发了一种更快速的流式细胞术为基础的突变检测与CHO AL细胞，使用抗CD 59单克隆抗体检测突变体，不需要colony formation.MethodsCHO AL细胞与γ射线或N-甲基-N′-硝基-N-亚硝基胍（MNNG）处理，然后允许生长不同的时间突变体的表达。用抗CD 59单克隆抗体标记细胞，并通过流式细胞术进行分析。将不同比例的CD 59阳性和阴性细胞混合，证明该试验具有高度线性（r2= 0.9999）和灵敏度（<0.05%背景突变体）。CD 59诱导突变体的产量与断裂剂（γ辐射）和点诱变剂（MNNG）的剂量呈线性相关。突变体产量是时间和治疗specific.ConclusionsMutations诱导的遗传毒性剂可以快速，灵敏地测量在CHO AL细胞使用流式细胞术。© 2005 Wiley利斯公司
BackgroundA sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO AL) that stably incorporates human chromosome 11. The assay measures mutations in theCD59gene on chromosome 11 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 ALcells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation.MethodsCHO ALcells were treated with γ‐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.ResultsNegative 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 (r2= 0.9999) and sensitive (<0.05% background mutants). The yield ofCD59‐inducible mutants was linearly related to dose for a clastogen (γ‐radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific.ConclusionsMutations induced by genotoxic agents can be rapidly and sensitively measured in CHO ALcells using flow cytometry. © 2005 Wiley‐Liss, Inc.