Measurement of cell proliferation by heavy water labeling
Measurement of cell proliferation by heavy water labeling
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DOI：
10.1038/nprot.2007.420
复制
发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Hellerstein, Marc K.
中科院分区：
文献类型：
--
作者：
Busch, Robert;Neese, Richard A.;Hellerstein, Marc K.
DNA复制几乎只发生在细胞周期的S期，代表了细胞分裂的简单生化指标。先前用于测量细胞增殖速率的方法具有重要的局限性。在这里，我们描述了用于测量细胞增殖和死亡率的实验方案的基础上，从重水（（H2O）-H-2）的氘（H-2）的掺入到DNA中的嘌呤脱氧核糖核苷酸的脱氧核糖部分的分裂细胞。通过气相色谱/质谱法测量标记掺入。对基本方案的修改允许分析小细胞样品（低至2，000个细胞）。本文介绍了有效利用这些方法测量体内细胞增殖率和死亡率的理论基础和操作要求。这些方法在人类中使用是安全的，与替代技术相比具有技术和解释优势，并且可以用于少量细胞。该方案能够在动物和人类中确定新分裂细胞的分数或绝对数量及其随后的存活动力学的体内研究。
DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium (H-2) from heavy water ((H2O)-H-2) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/ mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.