通过[3H]胸腺嘧啶核苷标记的DNA的选择性沉淀检测凋亡细胞。
Detection of apoptotic cells by selective precipitation of [3H]thymidine-labelled DNA.
作者信息
Patki A H, Lederman M M
机构信息
Department of Medicine, Case Western Reserve University School of Medicine, University Hospitals of Cleveland, Ohio 44106, USA.
出版信息
Clin Diagn Lab Immunol. 1996 Jan;3(1):1-4. doi: 10.1128/cdli.3.1.1-4.1996.
Apoptosis is characterized by systematic fragmentation of high-molecular-weight DNA into oligonucleosome fragments. A sensitive method for detection of apoptotic cells involving [3H]thymidine-labelled DNA is presented. Cells from mid-log-phase cultures were labelled with [3H]thymidine for 15 to 18 h and then exposed to gamma irradiation to induce apoptosis. A modified Hirt method was used to separate low-molecular-weight DNA from high-molecular-weight DNA. The percentage of fragmented DNA and high-molecular-weight DNA were measured by scintillation spectrometry. This method was compared with an established flow cytometric method for detection of apoptotic cells by using propidium iodide staining of DNA. We observed a good correlation between these two methods in detecting apoptosis. Hence, expensive flow cytometric assays for detection of apoptosis in dividing cells may be replaceable by a method involving [3H]thymidine labelling of DNA and separation of low-molecular-weight DNA from high-molecular-weight DNA by precipitation.
细胞凋亡的特征是高分子量DNA系统性断裂成寡核小体片段。本文介绍了一种检测凋亡细胞的灵敏方法，该方法涉及用[³H]胸腺嘧啶核苷标记DNA。对数中期培养的细胞用[³H]胸腺嘧啶核苷标记15至18小时，然后暴露于γ射线照射以诱导细胞凋亡。采用改良的赫特方法将低分子量DNA与高分子量DNA分离。通过闪烁光谱法测量断裂DNA和高分子量DNA的百分比。通过用碘化丙啶对DNA染色，将该方法与一种既定的流式细胞术检测凋亡细胞的方法进行比较。我们观察到这两种方法在检测细胞凋亡方面具有良好的相关性。因此，用于检测分裂细胞中细胞凋亡的昂贵流式细胞术检测方法可能可被一种涉及用[³H]胸腺嘧啶核苷标记DNA并通过沉淀从高分子量DNA中分离低分子量DNA的方法所替代。
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