University of Queensland Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, Brisbane, Australia.
Biotechniques. 2010 May;48(5):379-86. doi: 10.2144/000113410.
Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.
细胞增殖是生物体发育、组织更新以及癌症等多种疾病状态的基础。通过高通量筛选对增殖进行体外测量，可以快速描述小分子或基因治疗对原代和已建立细胞系的影响。目前直接测量细胞周期的方法不适用于高通量处理和分析。在这里，我们报告了通过将 5-乙炔基-2'-脱氧尿苷（EdU）掺入到高通量液体处理和高内涵成像分析中，对检测 DNA 合成的化学方法进行了适应性改造。我们证明，EdU 掺入的化学检测对于高内涵成像的高分辨率分析和 DNA 合成的定量是有效的。为了验证这个检测平台，我们用已知会影响细胞增殖的培养基补充剂和药理学抑制剂处理 MCF10A 细胞。用特定的激酶抑制剂处理表明，EGF 和血清刺激既使用有丝分裂原细胞外信号调节激酶（MEK）/细胞外调节激酶（ERK）信号网络，也使用磷酸肌醇-3 激酶（PI3K）/蛋白激酶 B（AKT）信号网络。如本文所述，这种方法快速、可靠且廉价，可产生易于解释的稳健数据。
