The bone marrow is the major hematopoietic organ, consisting of distinct microenvironmental niches for the production of hematopoietic cells. Advanced visualizing methods are required to define and better understand the interactions between stromal and hematopoietic cells. In this chapter, we describe an ex vivo whole-mount imaging technique of the bone marrow, which allows for a fast, high-quality, and three-dimensional visualization of different bone marrow components. We provide a guide for conducting adoptive transfer experiments of fluorescently labeled leukocytes and visualizing their location in the bone marrow with respect to the bone marrow vasculature. This method presents a quick, easy, and inexpensive approach to image the bone marrow in three dimensions.The soft marrow tissues, which are found disseminated throughout bone cavities, are prime sites for hematopoietic cell production, development, and control of immune responses, and regulation of skeletal metabolism. These essential functions are executed through the concerted and finely tuned interaction of a large variety of cell types of hematopoietic and nonhematopoietic origin, through yet largely unknown sophisticated molecular mechanisms. A fundamental insight of the biological underpinnings of organ function can be gained from the microscopic study of the bone marrow (BM), its complex structural organization and the existence of cell-specific spatial associations. Albeit the application of advanced imaging techniques to the analysis of BM has historically proved challenging, recent technological developments now enable the interrogation of organ-wide regions of marrow tissues in three dimensions at high resolution. Here, we provide a detailed experimental protocol for the generation of thick slices of BM from murine femoral cavities, the immunostaining of cellular and structural components within these samples, and their optical clearing, which enhances the depth at which optical sectioning can be performed with standard confocal microscopes. Collectively, the experimental pipeline here described allows for the rendering of single-cell resolution, multidimensional reconstructions of vast volumes of the complex BM microenvironment.Immunofluorescence is an indispensable method for the identification, localization and study of the expression of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections of human bone marrow. However, the procedure shows technical limitations because of the chemical and physical treatments required for sample processing before imaging. Here we describe a revisited protocol to obtain high-resolution images of human bone marrow trephine biopsies, improving the antigen-antibody recognition and preserving the morphology and the architecture of the bone marrow microenvironment.The intrinsic properties of self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) play a critical role in the regeneration of mature hematopoietic cells at steady state and in stress conditions including bleeding, inflammation and aging. Common techniques such as flow cytometry and genetic methods have answered many questions about their intrinsic and extrinsic regulation. Using these approaches, it was demonstrated that HSPCs in the bone marrow demonstrate low rates of proliferation and apoptosis. This dormant phenotype is associated with a low production of reactive oxygen species and low mitochondrial activity.Here, we describe the methodology to characterize the physiologic state of HSPCs isolated from their native hematopoietic organ using flow cytometry-based assays. buy CB-839 These protocols allow evaluation of their ROS levels and activated signaling pathways under various conditions.B cell development is a stepwise process encompassing several B cell precursor stages that can be phenotypically distinguished, and that is taking place in the bone marrow in adults. Interestingly, within the bone marrow B cell precursors coexist with the most differentiated actors of this lineage, the plasma cells. In this chapter, we describe a method to recover cells from the bone marrow and a flow cytometric approach to identify each subpopulation of the B cell lineage that resides within the bone marrow compartment. This protocol focuses on membrane markers to discriminate all the B cell subpopulations in order to preserve cell integrity during experimentation and for further analyses.Bone marrow stromal cells (BMSCs) account for an extremely small percentage of total bone marrow cells; therefore, it is technically challenging to harvest a good quantity of BMSCs with good viability using fluorescence-activated cell sorting (FACS). Here, we describe the methods to effectively isolate BMSCs for flow cytometry analyses and subsequent FACS. Use of transgenic reporter lines facilitates FACS-based isolation of BMSCs, aiding to uncover fundamental characteristics of these diverse cell populations.Flow cytometry has been widely used to detect a single event by means of multiparametric fluorescence measurements. Here we describe a method to analyze subsets of hematopoietic stem and progenitor cells isolated from long bones of mice. We further show that this method allows for comparing JAM-C protein expression between subsets of hematopoietic stem and progenitor cells.Bone marrow mesenchymal stromal cells (MSCs) play an essential role in the regulation of normal and leukemic hematopoiesis. Their multipotent potential of differentiation also makes them an interesting therapeutic tool. Among factors involved in the regulation of MSCs, energy metabolism plays a key role in their proliferation and differentiation. Seahorse Bioscience introduced extracellular flux technology to the life sciences market in 2006. This methodology allows, in living cells and in real time, the concomitant determination of basal oxygen consumption, glycolysis rates, ATP production, and respiratory capacity in a single experiment. Here we describe the protocol used to study concomitantly the respiratory and glycolytic metabolism of primary MSCs from the determination of oxygen consumption (OCR) and extracellular acidification (ECAR) rates.