Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).

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The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the

An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary