Busch Robert, Neese Richard A, Awada Mohamad, Hayes Gregory M, Hellerstein Marc K
KineMed Inc., 5980 Horton Street, Emeryville, California 94608, USA.
Nat Protoc. 2007;2(12):3045-57. doi: 10.1038/nprot.2007.420.
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 ((2)H) from heavy water ((2)H(2)O) 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.
DNA复制几乎只发生在细胞周期的S期,是细胞分裂的一个简单生化指标。以往测量细胞增殖率的方法存在重要局限性。在此,我们描述了基于将重水(²H₂O)中的氘(²H)掺入分裂细胞DNA中嘌呤脱氧核糖核苷酸的脱氧核糖部分来测量细胞增殖和死亡率的实验方案。通过气相色谱/质谱法测量标记掺入情况。对基本方案的修改允许分析小细胞样本(低至2000个细胞)。描述了有效使用这些方法测量体内细胞增殖和死亡率的理论基础和操作要求。这些方法在人体使用安全,与其他技术相比具有技术和解释优势,可用于少量细胞。这些方案能够对动物和人类中新分裂细胞的比例或绝对数量及其后续存活动力学进行确定性的体内研究。