Notes: The effect of E4F1 on metabolism was assessed by comparing metabolites in primary fibroblasts from E4F1-knockout and control mice. The NAD+/NADH ratio was measured with the NAD/NADH-Glo™ Assay. (4842)

Notes: Inhibition of HCT116 cell eEF2 kinase with the JAN-849 inhibitor lead to a dramatic increase in caspase-3/7 activity when the cells were cultured in medium without glucose for 24 hours. The increase could be blocked with cycloheximide (translation elongation inhibitor) or harringtonine (protein synthesis initiation inhibitor). Caspase activity was monitored with the Caspase-Glo® 3/7 Assay. A549 cells exposed to glucose-free medium had an increase in cytotoxicity that was partially abrogated by cycloheximide treatment. Knockdown of eEF2 kinase expression with siRNA treatment doubled the amount of cell death in glucose-free media, and cycloheximide treatment reduced cell death to about half. Cytotoxicity was monitored with the CellTox™ Green Cytotoxicity Assay using the end-point protocol. (4709)

Notes: Mouse embryonic fibroblasts were treated with vehicle or DPQ for 1 hour followed by exposure to H₂O₂.  Intracellular NAD+ was measured with the NAD/NADH-Glo™ Assay and cellular ATP was measured with the CellTiter-Glo® Viability Assay. (4845)

Notes: Nanoluciferase was used to characterize the conditions required to reliably perform single-cell BRET imaging of protein-protein interaction. HEK293T cells were cultured and transfected with vectors containing the Nluc gene sequence, such as is provided in the pNL1.1 Vector. Extracellular signal-related kinase (ERK) activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors, was detected by an Nluc-optimized BRET-based sensor of ERK activity. The authors report that the use of Nanoluciferase improved the resolution in terms of both time and space, enhanced the duration of signal stability, expanded the dynamic range and increased the sensitivity of the BRET signals in single-cell imaging. (4691)

Notes: Agonist antibodies targeting T cells and other immune cells to simulate immune activation have shown to be promising for cancer therapeutics. Here, the NanoBRET™ Protein-Protein Interaction Assay was used to measure hexamerization of anti-OX40 antibodies on the cell surface. Specific anti-OX40 antibody mutations were analyzed for increased antibody multimerization and engagement. Mutations promoting IgG hexamerization showed enhanced agonistic activity. (5056)

Notes: B16 F10 murine melanoma cells were treated with fenofibrate and the ratios of NADP+/NADPH and GSH/GSSG were examined after PPARa knockdown or over-expression. The NADP+/NADPH levels did not change appreciably but the GSH/GSSG levels did fluctuate. The study used the NADP/NADPH-Glo™ and GSH/GSSG-Glo™ Assays. Assays were read on a GloMax® 20/20 Luminometer. (4852)

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: Researchers set out to identify CBP/EP300 bromodomain inhibitors potent to both in vitro targets and targets in cellular target engagement assays. They developed a series of selective probes of CBP/EP300 bromodomains identified first by fragment screening. They next substituted and modified parts of the fragments to improve potency and selectivity.

To determine whether improvements in CBP bromodomain inhibition that were observed in their biochemical assay would translate to a cellular context, they used a bioluminescence resonance energy transfer assay, NanoBRET, where a small CBP/EP300 bromodomain inhibitor disrupted the interaction between a HaloTag-labeled histone and bromodomain conjugated to NanoLuc® Luciferase.

NanoBRET™ CBP/Histone H3.3 Interaction Assay (Cat.# N1850) and FuGENE® HD Transfection Reagent (Cat.# E2311) were used in the cell-based assay. (4717)

Notes: The effect of sirtuin2 overexpression on Leishmania infatum NAD+ homeostasis was assessed with the NAD/NADH-Glo™ Assay. (4844)

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: Both A549 and H460 cells were treated with the glutaminase inhibitor, BPTES. ATP content or viability was reduced about 30% in A549 cells and 60% in H460 cells. A549 and H460 cell ATP levels were mostly recovered by co-treatment with GSHE, a bioavailable form of glutathione. Despite a 60% decrease in ATP, H460 cells only demonstrated 5% cytotoxicity and A549 cells about 2.5% cytotoxicity. Co-treatment with GSHE reduced cytotoxicity to at or near background—no treatment levels. ATP content was assessed with the CellTiter-Glo® 2.0 Assay, and cytotoxicity was judged with the CellTox™ Green Cytotoxicity Assay. (4710)

Notes: In this experiment, cells were exposed to various concentrations of methylmercury, endosulfan, or 2,5-hexanedione for 24 hours. Then, the RealTime-Glo™ Assay was used to assess cell viability.  (4866)

Notes: This paper describes a new method for assessing receptor-mediated antibody internalization, a key mechanism underlying several anti-cancer antibody therapeutics. The method allows uses a hydrophilic, bright pH sensor dye (pHAb dye), which becomes fluorescent at acidic pH. Upon antibody binding to a receptor, the dyes conjugated to the antibody are not fluorescent due to the neutral pH of the media environment. Upon antibody internalization and trafficking into endosomal and lysosomal vesicles, the pH drops and dyes become highly fluorescent. The authors show the effectiveness of the method using two different therapeutic antibodies – Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR). (4633)

Notes: Human adipose-derived stem cells were cultured in the presence or absence of the Integra® collagen-based scaffold and monitored for proliferation over a 144-hour period. Cells were seeded with 1,000 cells using the RealTime-Glo™ MT Cell Viability Assay reagents and luminescence monitored every 24 hours. (4702)

Notes:   (5204)

Notes: RealTime-Glo™ MT Cell Viability Assay determined 3 days’ cell proliferation of the 3D printed constructs.  (4873)

Notes: The authors were investigating new methods of biofabrication of 3D multicellular constructs.  Cells were incorporated into a macroporous scaffold, encapsulated with alginate and printed onto a cold surface with agarose.  The viability immediately after printing was monitored hourly over 72 hours with the RealTime-Glo™ MT Cell Viability Assay. Total cell viability in the 3D constructs was monitored at day 2, 7 and 14 with the CellTiter-Glo® 3D Cell Viability Assay. (4819)

Notes: The rAAV-mediated genome editing technology was used to introduce a NanoLuc^® reporter sequence downstream of and in frame with the last coding exon of the HIF1A gene in HCT116 cells. This created a reporter cell line with a single allele of HIF1A endogenously tagged with a NanoLuc^® reporter fusion. The cell line was used to study compound inhibition of Hif1a signaling. NanoLuc^® luciferase activity was measured using Nano-Glo^® reagent. For qHTS, cells at 1500 cells/well in 1536-well plates were incubated with test compounds at 37°C, 5% CO₂, 1% O₂ for 18 hours in a humidified CO₂ incubator with variable oxygen control, followed by addition of Nano-Glo^® reagent or CellTiter-Glo^® cell viability assay reagent. The HRE-bla assay was conducted using the CellTiter-Glo^® reagent. HCT116 and ME-180 cell proliferation was quantified as relative luminescence unit (RLU) values using the CellTiter-Glo^® viability assay reagent. (4761)

Notes: Researchers were interested in screening compounds to inhibit endogenous class I and II histone deacetylases (HDACs). HCT116, HEK293, HepG2, and SU-DHL-6 cells were seeded at various cell densities (e.g., 1,500 or 4,000 cells/well) into 1,536-well white solid-bottom assay plates in a total volume of 5µl. Plates were treated with fibronectin prior to plating 5µl of human neural stem cells. Then 23nl of test compounds or DMSO were added to each well, and the plate incubated at 37°C for 1, 3, 6 or 18 hours. After incubation with the compounds, either the HDAC-Glo™ I/II Assay or CellTiter-Glo® Luminescent Cell Viability Assay were added to each well. Researchers confirmed 37 known HDAC inhibitors from two libraries of known epigenetics-active compounds and identified a group of potential HDAC inhibitors when screening the 2527 small-molecule compounds in the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection. (4697)

Notes: The study found that cells undergoing IKKβ-knockdown had reduced cellular NADPH levels as judged through use of the NADP/NADPH-Glo™ Assay. Data are presented in the supplement. (4857)

Notes: Several methods of making cryogels were evaluated for the ability of LNCaP cells to colonize and proliferate. LNCaP cell viability was monitored with the CellTiter-Glo® 3D Cell Viability Assay.  (4812)

Notes: SH-SY5Y cells were either plated above or incorporated into hydrogels containing an amyloid protein or collagen. The 3D cultures (incorporated into the hydrogels) viability was measured with the CellTiter-Glo® 3D Cell Viability Assay.  (4820)

Notes: The NAD/NADH-Glo™ Assay was used to monitor the NAD/NADH ratio of SKOv3 and SKOv3-ARHI cells with ARHI induction and glutaminase inhibition. (4836)

Notes: Several matrices (Matrigel, Cultrex Basement Membrane Extract and Alvetex plates) were evaluated for growth of DU145 and LNCaP cells in comparison to 2D culture. For DU145 cultures, Alvetex plates and 2D cultures were very similar in cell viability at 72 hours but 2D overwhelmed the 3D methods by 120 hours. The DU145 cells barely grew on the Matrigel or Basement Membrane Extract.   For LNCaP cells, all were similar at 72 hours but 2D was best at 120 hours with Matrigel and Alvetex plates at 50–60%.The different culture methods were also evaluated for compound toxicity. 2D cultures of DU145 were most susceptible to Docetaxel but mostly insensitive to rapamycin. Alvetex plates grown DU145 were insenstive to rapamycin but Matrigel and basement membrane extract showed some sensitivity.  LNCaP cells grown in all four conditions were susceptible to docataxel and rapamycin with 2D and Alvetex-grown cells being most susceptible. Cell viability in all four growth conditions was measured with the CellTiter-Glo® 3D Cell Viability Assay. (4813)

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)