Notes: The authors demonstrate that β-arrestin1 and β-arrestin2 differ in there interaction with PAC1R by using the Promega NanoBiT® technology. (5082)

Notes: The post-transcriptional modification of neddylation helps regulate protein activity, stability and localization. NanoLuc® Binary Technology (NanoBiT) was used to monitor covalent neddylation of Cul1 in a cellular context. SmBiT-Nedd8 and Cul1-LgBiT were expressed in HEK293 cells and luminescence was monitored as a response to a Nedd8-Cul1 covalent protein modification. Further, neddylation was monitored in the presence of both inhibitors and activators, and a concentration- and time-dependent response in luminescence was observed. (5076)

Notes: Proliferation assays were performed using the CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) and the GloMax^® Discover System. (5080)

Notes: Cyclin G associated kinase (GAK) is a central regulator of viral and bacterial host cell entry. The synthesis and characterization of selective and potent GAK inhibitors is described. Inhibitors were assessed for ability to compete for GAK binding using the NanoBRET Target Engagement system. Briefly, a BRET signal was observed with the NanoLuc-GAK fusion in the presence of a red-shifted dye specific to the GAK ATP binding site. Upon treatment with GAK inhibitors, the dye was competed away, and a loss of BRET signal was observed. (5130)

Notes: Cell viability was used as a reference parameter for acute cytotoxicity of airborne particulate matter and was assessed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. Luminescent signal was measured using the GloMax^® Discover System. (5085)

Notes: Lysyl Hydroxylase 3 is an enzyme involved in collagen synthesis that has both lysyl hydroxylase and glycosyltransferase activity. Lysyl hydroxylase activity can be measured using Succinate-Glo. In this citation the authors use both Succinate-Glo and UDP-Glo to measure the separate lysyl hydroxylase and glycosyltransferase activities of LH3. (5184)

Notes: The authors use Promega HiBiT and NanoBRET™ technologies to monitor PROTAC-mediated degradation. (5081)

Notes: The researchers demonstrate use of the NanoBRET™ Target Engagement Intracellular Kinase Assays to report on kinase target engagement in real time for quantitative inhibitor profiling of 178 kinases, including over 40 integral membrane receptors. (4936)

Notes: Researchers evaluated the impact of inhibiting autophagy on cells in vitro using cell viability, cytotoxicity and caspase activity assays. (5033)

Notes: Human PBMC were isolated and encapsulated in a alginate-collagen matrix (via electrostatic bead generator) either alone or after infection with Mycobacterium tuberculosis. Viability of PBMC-alone or M. tuberculosis-infected PBMC microspheres were measured with the CellTiter-Glo® 3D Cell Viability Assay. Luminescence measurements in the study were accomplished with either the GloMax® 20/20 Luminometer or GloMax® Discover Instrument. (4815)

Notes: Using CRISPR/Cas9 gene editing, HiBiT was tagged to the C terminus of HIF1α and several of its downstream transcriptional target proteins. The Nano-Glo® Lytic Detection System was used to quantify HiBiT-tagged endogenous protein levels, and the Nano-Glo® Live Cell Assay System was used for real-time detection in live cells. Bioluminescence was detected using the GloMax® Discover System. This study demonstrated the ability to efficiently tag endogenous proteins with HiBiT, allowing fast and sensitive quantification of the response dynamics in their regulated expression and covalent modifications. (4869)

Notes: Six mosquitos per sample were frozen in liquid nitrogen and ground in a basic pH lysis buffer then heated to remove NAD+ then neutralized before measuring NADH with the NAD/NADH-Glo™ Assay System. To examine the effect of HNF4 on VLCAD and 3KCT gene expression, the authors employed gel shift assays and luciferase assays of the promoters in pGL4.10[luc2] and control with pGL4.73[hRluc/SV40] transfected into Drosophila S2 cells. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument.  (4834)

Notes: Adenoma-derived organoids were established in Matrigel in 96--well plates at 10,000 cells per well. Cells were treated with a γ-secretase inhibitor or vehicle.  Growth of organoids was tremendously reduced compared to vehicle control after 5 days of treatment.  Cell viability was measured with the CellTiter-Glo® 3D Cell Viability Assay. Luminescence was measured with a GloMax® Instrument. (4814)

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag^® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

Notes: CRISPR/Cas9 editing was used to tag the GRP78 gene with NanoLuc^® luciferase in order to create a reporter of the intrinsic promoter activity of this gene. The authors used a single guide RNA against the N-terminal coding region of exon1 of the human GRP78 gene and constructed a donor gene that contains the puromycin-resistant gene following the NanoLuc^® gene. The NanoLuc^® gene was aligned with the puromycin-resistant gene through the 2A peptide sequence and was inserted into the pGL3-based vector (NL-2a-Puro-pGL3). To generate the donor gene, G78-NL, the N-terminal coding region that includes exon 1 of human GRP78 was inserted into the NanoLuc^® gene in the NL-2a-Puro-pGL3. After lysis of the cells expressing the indicated gene in 24- or 96-well plates with passive lysis buffer, the lysate was mixed with the NanoLuc^® substrate, and the luciferase activity in each well was measured using a GloMax^® luminometer. In some cases, the culture medium from each well was mixed with the NanoLuc^® substrate, and the activity for each was measured. (4754)

Notes: Proteins fused to NanoLuc^® luciferase were used as the energy donor and AlexaFluor conjugated nucleic acids were used as the energy acceptor in BRET assays that measured protein:nucleic acid interactions. The assay was demonstrated with immunopurified proteins, cell homogenates, and in whole cells. NanoLuc^® fusion protein construction, expression, and purification were performed using the vectors pFN31K Nluc CMV-neo for amino-terminal clones and pFC32K Nluc CMV-neo for carboxy-terminal clones. BRET assays were performed in white 96-well plates. Alexa-linked anti-sense oligonucleotides at the indicated concentrations were incubated at room temperature for 15 min in 1X binding buffer with 106 RLU/well of immunoprecipitated NLuc fusion protein or whole cell lysate. Following the incubation, NanoGlo^® substrate was added at 0.1 μl/well. Readings were performed using a GloMax^® Discover System. (4757)

Notes: 2D and 3D proliferation assays were performed using the CellTiter-Glo® Reagent with a GloMax® Discover System. (4745)

Notes: The glioblastoma cell lines U-251 MG (containing mutant p53) and U-87 MG (containing wildtype p53) were grown as microspheroids on ultra-low attachment 96-well plates and treated with monocarboxylate transporter (MCT) 1 inhibitors. Morphology was examined, and viability determined by transferring well contents to white 96-well plates and assaying with the CellTiter-Glo® 3D Cell Viability Assay. Luminescence was measured using a GloMax® Discover Instrument. Relative viability was compared to the same cells grown under monolayer conditions. Cells grown under 3D conditions were more susceptible to the MCT inhibitors. (4643)

Notes: ATP levels in lung secretions from CF patients were quantified by a luciferin-luciferase assay, using the GloMax^® Discover System. (4748)

Notes: The authors of this paper describe the application of a non-radioactive, bioluminescent assay to measure glucose uptake. The assay, now available as the Glucose Uptake-Glo™ Assay, showed similar sensitivity and produced comparable results to isotopic methods and is amenable to HTS. Additionally the authors performed the glucose-uptake assay in multiplex with the RealTime-Glo® MT Cell Viability Assay to obtain more information on the health of the cells. Luminescent assay results were measured using the GloMax® Discover Detection System. (4762)

Notes: Researchers were interested in detecting Helicobacter pylori infecting C57BL/6 mice in vivo using a fluorescent probe specific for the 16S rRNA gene. To test if the probe, Cy3_HP_ LNA/2OMe _PS, was cytotoxic to cells, the human gastric epithelial cell line AGS was seeded in a 96-well plate at 1.6 × 10⁵ cells/well and the next day, medium changed and 0.4–2 µM of probe or vehicle only was added to the AGS cells. After 24 hours, cell viability was determined using the CellTilter 96® Aqueous One Solution Cell Proliferation Assay. The potential genotoxicity of the H. pylori-specific probe was assessed using the VITOTOX® Assay, and the luminescent signal measured every 5 minutes over 4 hours using the GloMax® Discover System. (4749)

Notes: The NADP/NADPH-Glo™ Assay was used to look at variations in the level of total NADP+ and NADPH in primary porcine aortic endothelial cells with knockdown of von Willebrand Factor and the presence or absence of angiotensin II. The assay was read with a GloMax® Instrument. (4853)

Notes: Researchers were interested in the characteristics of nanoparticles that associated with proteins in blood. Nanoparticles (NP) were incubated with human plasma for 1 hour at 37°C, centrifuged, washed with PBS and resuspended before adding 10µl of NP suspension to a 96-well plate. The amount of protein was assessed using 150µl of Pierce protein assay reagent and absorbance at 660nm measured with the GloMax® Discover System. Cytotoxicity of NPs was tested by adding 10µg/ml of each NP formulation to HeLa cells in a 96-well plate. After 24 hours, MTT was added to assess cell viability, and the resulting 570nm absorbance measured using the GloMax® Discover System. (4750)

Notes: The bromodomain and extraterminal (BET) family of proteins contain two bromodomains. A probe compound, biBET, capable of binding both bromodomains of BET proteins in cis is characterized. BDR4-NanoLuc and Halo-tagged histone H3 fusions are used to monitor biBET binding with the NanoBRET Target Engagement system. Interestingly, bivalent binding lead to slower displacement of inhibitor from BDR4 and increased potency. (5078)

Notes: BEAS2B human bronchial epithelial cells were cultured for months in the presence of cigarette smoke condensate. The cells showed metabolic reprogramming including changes in the NADPH/NADP+ ratio. The measurement was made with the NADP/NADPH-Glo™ Assay and read on a GloMax® Instrument.  (4855)