Notes: The NADP+/NADPH ratio was monitored in two cell lines, one with a glycolytic profile, MB-231, and an oxidative profile, SiHa, to monitor the effects of 6-AN treatment. The ratio was used as a measure of the pentose phosphate pathway. The ratio was monitored with the NADP/NADPH-Glo™ Assay. (4848)

Notes: The effect of SMYD3 and ERK2 on hepatitis B RNA expression was examine in transfected Huh-7.5 human hepatoma cells. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the GoScript™ Reverse Transcription System followed by dye-based qPCR analysis. The effect of SMYD3 and ERK2 on AP1-dependent and NF-κB-dependent gene expression in the cells was examined with the pGL4.44 [luc2P/AP1 RE/Hygro] and pGL4.32 [luc2P/NF-κB-RE/Hygro] Reporter vectors using the Dual-Luciferase® Reporter Assay System with a pRL-TK Renilla luciferase control. Reporter assays were read with a GloMax® Instrument. (4603)

Notes: In this study, researchers used high-throughput compatible 384-well and 1536-well assay plate formats with spheroids of various cell types. Viability and apoptosis of monolayer and 3D tumor models was assessed. (5223)

Notes: One ml of 0.2 OD600 Mycobacterium tuberculosis (Mtb H37Rv) was treated with laterosporulin10 for 10 or 60 minutes then lysed with the assistance of a bead beater. The cleared lysate was analyzed for NAD/NADH and NADP/NADPH ratios using the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays. Both ratios fell dramatically upon treatment. (4827)

Notes: The receptor binding assays of the mature INSL5 and R3/I5 mutants were carried out using a NanoLuc-conjugated R3/I5 peptide as tracer and the human embryonic kidney (HEK) 293T cells transiently overexpressing human RXFP3 or human RXFP4 as receptor source. The receptor activation assays of the mature INSL5 and R3/I5 mutants were carried out using a cAMP-response element (CRE)-controlled NanoLuc^® reporter. Briefly, HEK293T cells were transiently cotransfected with the expression construct of human RXFP3 or human RXFP4 and the CRE-controlled NanoLuc^® reporter vector pNL1.2/CRE. (4756)

Notes: The authors demonstrated the use of the luminescent MTase-Glo™ Methyltransferase Assay for assaying the activity of a broad range of methyltransferases. Using protein and DNA methyltransferases with peptide, dsDNA oligos, mononucleosome and oligonucleosome substrates, methyltransferase reactions were performed in 384-well plates and activity determined by adding the two MTase-Glo™ Assay reagents and light detected using a luminometer. When the methyltransferase inhibitor GSK126 was used with the EZH2 complex in the MTase-Glo™ Assay, the IC₅₀ value was similar to that reported in the literature. The Z´ value generated using MTase-Glo™ with EHMT2-G9a and either a full length histone H3 or H3-derived peptide substrate was greater than 0.5. When screening the LOPAC library for potential MTase-Glo™ Assay inhibitors, few were found, showing the assay performed well in HTS applications. (4650)

Notes: Cell viability was assessed using the Real-Time-Glo™ MT Cell Viability Assay. (4865)

Notes: The effect of COUP-TFII on MPC1 promoter was evaluated with the Dual-Luciferase® Reporter Assay System. The effect of COUP-TFII on NADP+/NADPH levels in multiple prostate cancer cell lines was monitored with the NADP/NADPH-Glo™ Assay. The reduction in the ratio is likely due to the reduction in pentose phosphate shunt from lower glycolysis. (4847)

Notes: CellTiter-Glo® Cell Viability Assay was used to assess the vulnerability of myc-driven cancers to inhibitors of the NAMPT. Sensitivity was correlated to the level of myc expression. Apoptosis was assessed with the Caspase-Glo® 3/7 Assay. The NAD/NADH-Glo® Assay was used to provide a quantitative measure of NAD+ in the cells under study. (4835)

Notes: These authors designed a complementation reporter for detecting protein interactions under physiological conditions in mammalian cells. The engineered reporter (NanoBiT) is based on NanoLuc luciferase and is composed of two small subunits (18kDa and 1.3kDa), which associate weakly. The formation of a luminescent signal is dependent on the interaction characteristics of the proteins onto which the subunits are attached. The paper demonstrates utility of the NanoBiT assay for detecting protein interactions in live cells using  FRB/FKBP as an example, shows that response dynamics are rapid and reversible, and also illustrates use of the system to measure the pharmacology of kinase inhibitors that induce interaction between BRAF and CRAF. (4585)

Notes: This article details the advantages of NanoLuc® Luciferase in the scientific community. (4924)

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 authors site-specifically conjugated the NanoLuc^® luciferase reporter to WT and mutant FGF2 proteins to create non-radioactive tracers that could be used to quantify ligand-receptor interactions with fibroblast growth factor receptors. Prior to Nanoluc^® conjugation, the biological activities of recombinant WT and mutant FGF2s were measured using receptor activation potency assays using a serum response element (SRE)-controlled NanoLuc^® reporter (pNL1.2/SRE) in HEK 293T cells expressing endogenous FGF receptor. Receptor-binding assays using the FGF2-Nanoluc^® tracers were carried out on overexpressed and endogenous FGF receptor in HEK293T cells. (4758)

Notes: A NanoLuc^® luciferase reporter was used to further understand the mechanism of Nrf2 inhibition of proinflammatory cytokine gene induction. Approximately 1 kb upstream region of the IL6 gene TSS containing the WT ARE (GCTGAGTCA) or mutated ARE (AGATCTGAC) was conjugated to the translation start site of the NanoLuc^® gene in the pNL2.2 vector. 293T cells (2.0 × 10³ cells per well) were plated in 96-well plates 24 h before transfection. The NanoLuc^® reporter vectors were co-transfected with pQC-FLAG-hNRF2T80R (constitutively active NRF2) plasmid33 and pcDNA3-p65 plasmid. After 48 hours of the transfection, the luminescence was quantified and normalized using NanoGlo^® Dual-Luciferase^® Reporter Assay. (4752)

Notes: The NAD/NADH-Glo™ Assay was used to assess the effects of oxaloacetate, malate with or without glucose present on NAD+/NADH levels in SH-SY5Y cells at 20,000 cells per well. (4838)

Notes: The authors amplified the HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1 and DPA1 loci by PCR. The amplicons were then quantified with the QuantiFluor® dsDNA System prior to library preparation and Illumina (MiSeq) sequencing. (4799)

Notes: Normal human dermal fibroblasts were allowed to form microspheres. Eight microspheroids were seeded per well of a 24-well plate containing electrospun polyurethane. Microspheroids were also placed into wells of a standard tissue-culture-treated plate to serve as a positive control for 100% viability. Cells were cultured for 24 or 72 hours, incubated with CellTiter-Glo® 3D Reagent, and supernatants transferred to a 96-well plate for detection in a 96-well luminometer. Microspheroids cultured on the polyurethane were judged to be 95% viable. (4641)

Notes: Asparaginase treatment caused a significant increase in the NAD+/NADH ratio in all cell lines (data in supplement). The dinucleotide ratio was determined with the NAD/NADH-Glo™ Assay. (4839)

Notes: The effect of PHGDH knockdown on redox homeostasis in human breast cancer cell lines was examined with regard to GSH/GSSG ratios and NADPH levels under normoxic and hypoxic conditions. The study used the NADP/NADPH-Glo™ and GSH/GSSG-Glo™ Assays. (4850)

Notes: The authors utilized phosphoproteomics to identify host factors that are activated upon Influenza A infection. In total, 1116 proteins showed changes in regulation including cyclin-dependent kinases. The authors tested the effects of cyclin-dependent kinase inhibitors on cell viability and caspase activation using the CellTiter Glo® and Caspase-Glo® assays. (5154)

Notes: Proliferation assays were performed using the CellTiter-Glo^® Luminescent Cell Viability Assay and the GloMax^®-Multi+ Microplate Reader. (5083)

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: The authors used the CellTiter-Glo® Cell Viability Assay and CellTox™ Green Cytotoxicity Assay to monitor the reproducibility of their acoustic dispensing. The authors report that adding CellTox™ Green Cytotoxicity Assay at plating "gives more solid signals than adding at the end of the assay” and speculate that the dye stabilizes the DNA of dead cells. The CellTiter-Glo® Assay data supports that the dye is nontoxic in the 72-hour incubation. The authors routinely predispense 12.5nl of CellTox™ Green with preplated compounds into 1536-well assay plates using an Echo 550 device and store plates under nitrogen gas in storage pods to produce assay-ready plates awaiting cell additions. The authors also report preliminary results of using the nonlytic RealTime-Glo® Viability Assay and find highly correlated results with the lytic CellTiter-Glo® Assay. The authors relate that use of the nonlytic RealTime-Glo® and CellTox™ Green Assays open the possibility of doing further work with the same cells. (4703)

Notes: The CellTiter-Glo® 3D Cell Viability Assay was used to monitor viable MDA-MB-231 or MMTV-myc cells grown on Matrigel in 96-well plates. (4822)

Notes: A triple drug treatment-parthenolide, 2-deoxyglucose, and temsirolimus (PDT); each chosen for the ability to inhibit the pentose phosphate pathway and the Nrf-2-mediated anti-oxidant reponse-was tested against AML cells, leukemic stem/progenitor cells and normal heamtopoietic counterparts. The PDT treatment was potently toxic to the AML and leukemic stem/progentor cells with little toxicity to the normal cells. The NADPH and GSH/GSSG ratios were monitored in this study with the NADP/NADPH-Glo™ Assay and GSH/GSSG-Glo™ Assay.  (4856)