除了经典的B-DNA构象外，DNA还可以折叠成不同的次级结构。其中包括G-四链体结构（G4）。G4结构非常稳定，可以在DNA和RNA的特定富含鸟嘌呤区域中折叠。不同的体外、体内和体内实验已经证明，到目前为止所有测试的生物中都形成了G4结构。在高等真核生物中有超过700,000个预测的G4，在同一时间内可能不会全部形成。它们的形成受蛋白质动态调节，是细胞类型特异性的，甚至在细胞周期或不同外源或内源刺激（如感染或发育阶段）中也会改变G4水平。已经证明G4在癌细胞中积累，这些G4结构导致基因表达变化和肿瘤的突变负担。目前论述将特定靶向G4结构以影响癌基因表达作为抗癌治疗的方法。在肿瘤微环境中，不仅肿瘤细胞将成为G4稳定的靶点，还有诸如巨噬细胞等免疫细胞。尽管在多种生物和不同细胞类型中检测到G4，但我们对它们在免疫细胞中的作用了解甚少。在这里，我们提供了一份详细的方案，通过显微成像来检测脊椎动物和无脊椎动物巨噬细胞核中的G4形成。© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.