Notes: These researchers screened for microbiome metabolites that activated inflammasomes, using cell viability and caspase-1 activity assays, and a CRISPR genome-wide screen.  (5205)

Notes: This Assay Guidance Manual provides a resource to scientists optimizing assays used in drug discovery and development. The cytotoxicity chapter outlines several assays to detect dead cells, including LDH-Glo™, CytoTox-Glo™ and CellTox™ Green Cytotoxicity Assays, CellTiter-Glo® Luminescent Cell Viability Assay, CytoTox 96® Non-Radioactive Cytotoxicity Assay and CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (5198)

Notes: Researchers generated novel glioblastoma 3D cell models to better predict drug efficacy in patients. Two cell lines were used for high-throughput screening, where cell viability and apoptosis assays were used. (5224)

Notes: BDCRB selectively disrupts DNA packing in cytomegalovirus, a process which is critical for herpesvirus life-cycle. In this paper, the authors use guinea pig cytomegalovirus as a model for human infection and derive a resistant virus to characterize the BDCRB mechanism of action. Recombinant viruses containing an expression cassette for NanoLuc luciferase in either a wild-type or L406P mutant cytomegalovirus background were constructed. Luciferase activity was used as a proxy for viral yield in BDCRB treated or untreated cells. The L406P mutation did not show significant resistance to BDCRB treatment.  (5108)

Notes: The methodology of the 3D cell culture system, TRACER (tissue roll for analysis of cellular environment and response) to monitor cell phenotypes in hypoxic conditions is explained. Cell viability in response to drug treatment can be measured using the CellTiter-Glo® Luminescence Cell Viability Assay. (5183)

Notes: These authors evaluated the toxicity and the anti-inflammatory effects of various traditional herbal remedies used for asthma, with the goal of selecting promising candidates for further characterization. Remedies were tested for toxicity on THP-1 derived macrophages using the CellTiter-Glo® Luminescent Cell Viability Assay. The Caspase-Glo® 1 assay was used to evaluate inflammatory effects.

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: The role of mechanical properties of cell culture conditions in diabetes-linked cardiac dysfunction was evaluated. ATP levels were measured using the CellTiter-Glo® Luminescence Cell Viability Assay and a GloMax® instrument. Caspase activation and glucose uptake were determined with the Caspase-Glo® 3/7 and Glucose Uptake-Glo™ Assays. Cell growth environments closely matching the stiffness of cardiac muscle led to increased glucose susceptibility. (5167)

Notes: These authors used the Caspase-Glo® 3/7 and CellTiter-Glo® Assays in studies to determine the cytotoxicity of  34 small molecule kinase inhibitors in primary rat and human hepatocytes. (5009)

Notes: The role of USP6NL in metabolic rewiring in breast cancer was investigated. The CellTox™ Green and CellTiter-Glo® assays were used to measure cell toxicity and ATP levels, respectively. Depletion of USP6NL in certain breast cancer cell lines lead to decreased glucose uptake as measured by the Glucose Uptake-Glo™ assay. USP6NL was shown to be involved in activation of glycolysis in breast cancer cells. (5150)

Notes: Cell toxicity and ATP cell content were evaluated with the CellTox™ Green Cytotoxicity Assay and CellTiter-Glo® Luminescent Cell Viability Assay. Glucose uptake was measured with the Glucose Uptake-Glo™ Assay. (5004)

Notes: This paper details one example of using NanoLuc luciferase to create reporter viruses. NanoLuc-containing infectious particles for various types of flavivirus (e.g., Dengue, Zika, West Nile) were used in neutralization assays with serum samples following either infection or immunization. (5129)

Notes: The ten most common human liver cytochrome P450 enzymes were introduced into a HEK293T cell line to support the creation of high throughput screening assays. Luminogenic substrates for the P450 enzymes were used to assess activity. (5212)

Notes: Mutations in RAS are commonly associated with pancreatic, colorectal and non-small-cell lung cancers. Treatments targeting RAS in cancers have proven to be difficult due to compensatory resistance mechanisms and upstream pathway insensitivities. Here, SHP2, a protein linking receptor tyrosine kinase signaling to the MAS-RAF-MEK pathway, is shown to lead to cell senescence under growth factor-limiting conditions. The Beta-Glo^® Assay System is used to monitor cell senescence using senescence-associated β-galactosidase, and the CellTiter-Glo^® assay is used to determine cell viability. Together, inhibition of SHP2 showed downstream effects on the RAS signaling pathway and could be sufficient for tumor senescence. (5118)

Notes: These authors investigated silicon dioxide nanoparticles (SiNPs) as a potential cancer treatment and identified the mechanism mediating cancer cell death. The Caspase-Glo® 3/7, Caspase-Glo® 9, CellTiter-Glo® luminescent cell-viability, ROS-Glo™, and Caspase-Glo® 1 assays were used to monitor cellular response to SiNPs. Together, SiNPs were identified to activate the intrinsic apoptosis pathway and lead to cell death.

  (5165)

Notes: These authors used the CellTiter-Glo® and ViralTox-Glo® assays to screen a library of compounds for antiviral activity against Ebola and other flaviviruses. They initially performed screens in 384-well plates using a reporter-based assay for viral replication and CellTiter-Glo® to assess cytotoxicity. Having identified a promising compound, they then performed various dose-response curves to confirm that the dose that most effectively inhibited viral replication was not toxic to the host cells. The authors then confirmed that the compound was effective in assays using live viruses. For these assays they used the ViralTox-Glo® assay to assess effectiveness. (4939)

Notes: These authors tested various cell lines for propagation of Zika virus in vitro. Having identified the best cell lines, they performed dose-response experiments with known antiviral compounds, using CellTiter-Glo® to assess cytotoxicity and confirm that candidate compounds were effective in blocking viral replication while having minimal effect on the host cells. The authors also used ImProm-II reverse transcriptase™ and RNasin® ribonulease inhibitor in qRT-PCR assays performed as an alternative to plaque assays for quantifying viral titer. (4940)

Notes: The CellTiter-Glo^® assay was used in a high-throughput drug screening assay to identify compounds which increase mitochondrial ATP production in muscle cells. Hesperetin was found to both increase mitochondrial function and reduce oxidative stress as measured by the GSH/GSSG-Glo™ assay. The authors present a novel screening platform to identify possible therapeutics for sarcopenia and other mitochondrial dysfunction diseases. (5177)

Notes: Human cultured cells were plated at 1,000 cells/well in 100µl of medium in 96-well plates. The next day, the growth medium was replaced with fresh medium or medium containing 4mM oxaloacetate or aspartate. After 3 days, cell viability was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay. To assess the ratio of NADPH to NADP+, cells were lysed, mixed with the NADP/NADPH-Glo® Assay reagents and luminescent measured after 30 minutes. (4948)

Notes: Sorafenib is a small-molecule inhibitor that inhibits multiple kinases in vitro, These authors investigated the antitumoral effect of combining sorafenib with the alkylating agent cyclophosphamide in a breast cancer model (orthotopic 4T1-12B cells). They used the Caspase-Glo® 3/7 and CellTiter-Glo® Assays to assess apoptosis and cell viability, respectively. (5048)

Notes: Human primary hepatocytes at a density of 2 × 10⁴ were seeded into 96-well collagen-coated plates, incubated overnight and then stimulated with two compounds. After 48 hours, the levels of NAD+ was assessed using the NAD/NADH-Glo™ Assay. HepG2 and AML12 cells (5 × 10³) were treated with test compounds for 4 hours in a 384-well plate. Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay and cell necrosis evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (4953)

Notes: The role of the carcinogen Ochratoxin A in cellular inflammation was examined. HEK293 cells were treated with Ochratoxin A and cellular inflammatory markers were monitored. ATP levels and caspase activation were analyzed using the CellTiter Glo® and Caspase-Glo® assays. (5155)

Notes: Spheroid cultures were created from primary hepatocytes by the use of magnetic nanoparticles and magnetization. Hepatocyte spheroid cultures were treated with CYP-inducing or -inhibiting drugs for 72 hours. The media was removed and spheroid cell cultures washed, then luciferin pro-substrates (Promega) added, with the substrate used dependent on the treatment. After a 2-hour incubation, media was removed and aliquoted to new white-walled plates, and an equal volume of Luciferin Detection Reagent was added. At the same time, CellTiter-Glo™ Cell Viability Reagent was added to the original plates in an equal volume. All plates were incubated for 20 minutes before reading in a plate-based luminometer. (4872)

Notes: The development of NanoLuc Luciferase has led to a need for selective NanoLuc inhibitors to allow for bioluminescent suppression and multiplexing compatibility with existing assays. A lead compound with an IC50 of 600 nM against NanoLuc was further derivatized to create a family of potent and cell permeable inhibitors. These compounds were additionally developed to generate a second class of cell impermeable inhibitors. These inhibitors were tested for selectivity against Firefly luciferase and the NanoBiT system with no cross-reactivity.  (5106)