Prior to imaging, media was removed and replaced with 2?ml HBSS and the dish was placed on the heated stage of the LV200 for 15?minutes at 37C. targeted nanobodies can be utilized to probe receptor pharmacology. (Kalatskaya et?al., 2009). This, in addition to its short serum half-life (Hendrix et?al., 2000), has necessitated the development of more selective, long-lasting antagonists. The improved selectivity and extended half-lives of antibodies compared with small molecules has meant there has been much interest in using antibody-based approaches to target CXCR4 therapeutically (Hutchings et?al., 2017; Bobkov et?al., 2019). This has included the recent development of a panel of single-domain antibody fragments, called nanobodies, which are able to bind CXCR4 (Jahnichen et?al., 2010; de Wit et?al., 2017; Bobkov et?al., 2018; Van Hout et?al., 2018). Nanobodies are small proteins (circa 12C15?kDa), derived from the single variable fragments (VHH) of heavy-chain-only antibodies found in members of the Camelidae family. Nanobodies are known to be excellent conformational sensors due to their small size and three-dimensional structure (De Genst et?al., 2006). Furthermore, their elongated complementary determining region 3 (CDR3) enables nanobodies to engage hidden cavities and conformational epitopes (De Genst et?al., 2006). Nanobodies have been extensively used within the GPCR field to stabilize specific receptor conformations for crystallization (Rasmussen et?al., 2011; Ring et?al., 2013; Kruse et al., 2013; Huang et?al., 2015; Che et?al., 2018) and to elucidate new Oxybutynin conformational says (Staus et?al., 2016). This has also led to the development of these nanobodies as biosensors to investigate GPCR signaling (Irannejad et?al., 2013; Staus et?al., 2014, 2016; Stoeber et?al., 2018). The nanobodies used in these studies generally target intracellular regions of the GPCR, often binding in the same pocket as G proteins to act as G protein mimetics (Rasmussen et?al., 2011; Staus et?al., 2014; Stoeber et?al., 2018). Nanobodies that bind to the extracellular domains of GPCRs are Oxybutynin able to modulate receptor activity and business (De Groof et?al., 2019a). Several studies have investigated the therapeutic Oxybutynin potential of extracellular nanobodies that target chemokine GPCRs, including CXCR2 (Bradley et?al., 2015), CXCR4 (Jahnichen et?al., 2010; de Wit et?al., 2017; Bobkov et?al., 2018; Van Hout et?al., 2018), ACKR3 (Maussang et?al., 2013), and US28 (De Groof et?al., 2019b). Given their relatively large N terminus compared with the other class A GPCRs and the fact that their endogenous ligands are peptides, chemokine GPCRs are ideal candidates to target with extracellular nanobodies. Most recently, several nanobodies binding to the N terminus and second extracellular loop (ECL2) of CXCR4 were generated (Bobkov et?al., 2018; Van Hout et?al., 2018). For example, VUN400 was one of these nanobodies that acted as an antagonist and inhibited CXCL12-induced signaling by CXCR4, as well as internalization. eNOS Interestingly, VUN400 also showed a decreased potency of inhibiting CXCR4-mediated HIV-1 entry compared with its ability to inhibit CXCL12-induced signaling, suggesting a conformational sensitivity of the nanobody (Van Hout et?al., 2018). The recently developed NanoLuc binary technology (NanoBiT) splits the bright NanoLuc luciferase into two segments at the C-terminal region, the 18-kDa fragment (termed LgBiT), and the 1.3-kDa small complementation tag (termed SmBiT; Dixon et?al., 2016). These fragments have low intrinsic affinity and complement to form the full luminescent NanoLuc protein but with a reduced luminescence compared with the full-length NanoLuc (Dixon et?al., 2016). Other small complementary peptides with a range of affinities for LgBiT have been identified, including an 11-amino-acid sequence with very high affinity, termed HiBiT. The complemented HiBiT-LgBiT protein showed a luminescence output similar to that of the full-length NanoLuc, making it an ideal system to study proteins expressed at endogenous levels (Schwinn et?al., 2018). NanoBiT has been used to monitor protein-protein interactions, including GPCR oligomerization (Botta et?al., 2019), and the recruitment of G proteins and -arrestin to GPCRs (Hisano et al., 2019; Laschet et?al., 2018; Storme et?al., 2018), with the rapid complementation and maturation rate of the split NanoLuc luciferase enabling kinetic measurements. These studies made use of GPCRs with NanoBiT fused to their C-terminal domains. In addition, we have recently demonstrated the use of N-terminally-fused NanoBiT to monitor adenosine A1 receptor internalization (Soave et?al., 2019b), showing it was Oxybutynin possible to use NanoBiT to.