Cell Biology, University of Bern; Department of life sciences and medicine, University of Luxembourg;
Cell Biology, University of Bern.
J Vis Exp. 2021 May 4(171). doi: 10.3791/62642.
In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S>G2/M (progression from S to G2/M), and M phases.
在研究调控有丝分裂和 DNA 复制的基因时，通常会进行体内细胞周期进程分析。5-乙炔基-2'-脱氧尿苷 (EdU) 已被用于研究复制/S 期进展，而磷酸化组蛋白 H3 的抗体已被用于标记有丝分裂核和细胞。两种标记物的组合可将 G0/G1（间隙期）、S（复制期）和 M（有丝分裂期）区分开来，是评估有丝分裂基因敲低或缺失突变体对细胞周期进程影响的重要工具。然而，用于标记 EdU 标记细胞的试剂与几种二级抗体荧光标签不兼容。这使得免疫染色复杂化，其中使用初级和标记的二级抗体来标记 pH3 阳性有丝分裂细胞。本文描述了在果蝇幼虫神经干细胞中双重标记 EdU 和 pH3 的分步协议，该系统广泛用于研究有丝分裂因子。此外，还提供了用于图像分析和定量的方案，将标记的细胞分配到 3 个不同的类别，G0/G1、S、S>G2/M（从 S 期到 G2/M 期的进展）和 M 期。
