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Notably, these steps do not contribute to static assays in vitro. However, static transmigration assays are useful as negative controls in experiments testing the effects of shear stress on cell survival and function, including the ability of EC to support low affinity interactions (rolling) and firm adhesion. The choice of medium for experiments in the 3D device is important. Media contain different growth factors that may influence the survival of circulating cells, particularly during long incubation times. In addition, the concentrations of Ca2+ and Mg2+, which may influence adhesive interactions under conditions of physiological flow, vary considerably in commercial culture media. Other technical details that should be taken into account when planning experiments with the 3D device are discussed below. Selection of the EC Ku-0059436 research buy monolayer The cell surface signature of EC is influenced by various parameters, including the species and tissue of origin and the cell culture conditions, which should be taken into account in the experimental design. Some commonly used EC include lung-derived microvascular EC (LDMVEC), bone marrow-derived EC (BMDEC), brain-derived EC (BDEC), and HUVEC. The properties of EC also vary with their origin. For example, BMDEC constitutively express selectins and VCAM-1, which are responsible for rolling and adhesion, whereas BDEC, LDMVEC, and HUVEC do not express these molecules under normal conditions. Therefore, inserts coated with BDEC, LDMVEC, and HUVEC can be pretreated with TNF �� (10 ng/ml, 4 hr) or other factors to mimic inflammation and induce expression of homing molecules. Testing crosstalk with the local microenvironment There is growing interest in understanding how the local microenvironment regulates the functions of EC and participates in cell extravasation. Our results demonstrate that insertion of a monolayer of stromal cells beneath the EC monolayer significantly increases extravasation of circulating cells. This finding demonstrates that crosstalk between EC and stroma enhances the ability of the EC to support extravasation of circulating cells. Moreover, the insertion of cells from different microenvironments (e.g. mesenchymal stem cells, lung fibroblasts, tumor cells, astrocytes) could help to mimic specific vascular beds. In particular, a combination of brain-derived EC and astrocytes could be a useful approach to mimic the blood-brain barrier. Similarly, cells obtained from patients, or normal cells manipulated in vitro, could help mimic a specific diseased microenvironment. In our studies, we used lower inserts with stromal cells grown to 50% confluence. Although the optimal density of microenvironmental cells on the inserts will depend on the goals of the study, this can be readily manipulated and controlled.