1/20/2024 0 Comments Nucleo celular![]() Human trophoblast organoids (TOs) are a three-dimensional ex vivo culture model that can be used to study various aspects of placental development, physiology and pathology. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. ![]() Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. 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. 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. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Among them are G-quadruplex structures (G4s). In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. ![]() Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. ![]()
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