Notch Ligand Binding Assay Using Flow Cytometry

Notch signaling is an evolutionary conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.