Introduction S. aureus is a major cause of morbidity globally, and in the United States it contributes significantly to both hospital admissions and in-hospital morbidity [1, 2]. The increasing incidence of antibiotic-resistant strains increases the urgency of understanding the mechanisms by which this infection exerts its toxic acute effects, as well as potential longterm impact on infected patients, especially those with comorbid conditions. The major virulent toxin secreted by S. aureus is α-hemolysin (Hla). A Disintegrin And Metalloproteinase domain-containing protein-10 (ADAM10), which is involved in ectodomain shedding, is the eukaryotic receptor for Hla [3-5], and mediates vascular injury caused by Hla [6]. Almost all isolates of S. aureus express Hla, including methicillinresistant strains [7]. Recently, Hla has been shown to mediate VE-cadherin degradation in endothelial cells (EC) via ADAM10, affecting permeability [6]. Importantly, the Notch1 and 2 receptors are known ADAM10 targets [8]. Notch proteins are highly evolutionarily conserved. In mammals, the Notch pathway is comprised of the Jagged and Delta-like ligands, and the receptors Notch1 through Notch4. Both ligands and receptors are membrane-bound: in order for activation to take place, the ligand and receptor must be expressed in adjacent cells. Notch ligands are cleaved at Site 1 (S1) and can be post-translationally modified by glycosyltransferases, such as Fringe. Upon

The family of Notch proteins plays a key role in cell fate determination. Additionally, Notch proteins regulate critical functions of the endothelium, as well as other recruited supporting cells, in concert with other pathways. Despite significant advances in the field and extensive studies focused on elucidating this pathway, many questions remain regarding Notch activation and its upstream/ downstream effects, with vascular biology constituting one area of particular interest. Here, we provide a brief description of the components and functions of the Notch pathway in vasculature, followed by a detailed compilation of recommended methods of evaluation in vitro and in vivo. We provide a rationale for key elements when choosing different approaches and controls, strengths and limitations, and essential considerations when providing a meaningful interpretation of results. Our aim is to describe a careful approach to assessing Notch function in endothelial cells, based on underlying principles, with the overall goal of obtaining physiologically relevant information that will enhance our understanding of this pathway and its role in vascular biology.