Department of Biochemistry, Albert Einstein College of Medicine.
Department of Biochemistry, Albert Einstein College of Medicine;
J Vis Exp. 2022 May 5(183). doi: 10.3791/63606.
Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.
哺乳动物细胞的平面培养是一种广泛用于理解细胞生理学的体外方法，但由于细胞的异常快速复制，该系统在模拟实体组织方面受到限制。当模拟成熟染色质时，这尤其具有挑战性，因为快速复制的细胞经常参与 DNA 复制，并且具有异质的多倍体群体。下面介绍了一种使用三维（3D）细胞培养系统对静止染色质修饰进行建模、处理和分析的工作流程。使用该方案，肝癌细胞系在提供主动营养扩散和低切变力的培养箱中生长为可重复的 3D 球体。用丁酸钠和琥珀酸钠处理分别诱导组蛋白乙酰化和琥珀酰化的增加。组蛋白乙酰化和琥珀酰化水平的增加与更开放的染色质状态相关。然后收集球体以分离细胞核，从核中提取组蛋白以分析其翻译后修饰。通过与串联质谱在线连接的液相色谱法进行组蛋白分析，然后通过内部计算管道进行分析。最后，展示了用于研究组合组蛋白标记的频率和出现的数据分析表示的示例。
