Notes: Synthetic amyloid fibrils were created from α-synuclein and amyloid-β peptide 40. Fragment fibrils were made by freezing and sonicating the created fibrils. In vitro experiments suggested that fragment fibrils would interact and damage cellular membranes. This was confirmed by treating SH-SY5Y with both intact and fragmented fibrils over a 72-hour period. Cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay using the “zero step” addition to the cells at plating. Cytotoxicity was measured in the same wells at 24, 48 and 72 hours. (4714)

Notes: These authors describe a new method for monitoring ligand binding to GPCRs on the surface of living cells. They used NanoLuc luciferase in a BRET-based assay to detect the interactions of ligands with the receptors with an N-terminal NanoLuc fusion. (4589)

Notes: The authors of this study sought to determine whether cucurbitacin B has antitumor activity against the ovarian cancer cell line A2870 and whether it can sensitize the cisplatin-resistant cell line A2780CP to treatment. They compared caspase activity in A2780CP cells either preincubated with cucurbitacin B before treatment with cisplatin or cells not pretreated using the Caspase-Glo® 3/7 Caspase Assay. Oxidized and total glutathione were measured from both cell lines (before and after cisplatin exposure, with and without preincubation with cucurbitacin B) using the GSH/GSSH-Glo™ Glo Assay. The level of reactive oxygen species in cell lines was also measured by detecting ROS converted to H₂O₂ using the ROS-Glo™ Assay. Cell Viabilty of 3D spheroids formed from the A2780CP cell line was assessed using the CellTiter-Glo® 3D Cell Viability Assay. (4581)

Notes: In order to investigate the interaction of the Cullin-RING E3 ubiquitin ligase with Elongin A due to toxic damage to DNA, the authors chose a FRET assay consisting of mCherry-labeled Cullin-RING3 and HaloTag-labeled Elongin A (clone obtained from Promega as pFN21-TCEB3 then subcloned into another expression vector). Interaction of the two proteins was examined in UV irradiated HeLa and U2OS cells. As a control, a HaloTag Protein expression vector alone was used. Intracellular HaloTag-labeled protein visualization was accomplished with the HaloTag® R110Direct™ Ligand. Both cell lines were transfected at 50-60% confluency with 700ng of plasmid in glass bottom 35mm dishes. FuGENE® HD Reagent was used for HeLa cells and ViaFect™ Reagent was used for U2OS cells. (4674)

Notes: The authors describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. They monitored cell health for 72 hours from the same test samples, distinguished differential cell growth, and investigated drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements enabled them to detect cell death immediately (>75% signal decrease within 15 minutes of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects.They then screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points. (4590)

Notes: These authors describe use of firefly and NanoLuc® luciferases in a coincidence reporter system to screen compounds for their ability to activate transcription of the Parkin gene (PARK2), a potential target for treatment of Parkinson's disease. In a coincidence reporter system, a single transcript containing two luciferase genes is generated. Inclusion of a 2A “ribosomal skipping” sequence between the two luciferase genes enables translation of two reporter proteins. This approach is useful in drug screening because the use of two independent reporters to monitor a single transcriptional event provides a way to distinguish true “hits” from experimental artifacts caused by interaction between library compounds and reporter proteins. True “hits” (compounds that affect transcription of the gene being monitored) cause a signal from both reporters; false positives caused by interaction of compounds with the reporter protein cause a signal from only one reporter. This paper describes the first use of firefly and NanoLuc® luciferases, and the Nano-Glo® Dual-Luciferase® Reporter Assay, in a coincidence reporter system to screen a compound library in an HTS assay. (4565)

Notes: Differentiated L6 myocytes were seeded at 1 × 10⁵ cells/well in a 24-well plate containing a 4:1 ViaFect™ Transfection Reagent: DNA ratio containing 1.025µg of DNA (reverse transfection). Cells were transfected with 500ng of a pGL4.12 [luc2CP]-based Ucp3 promoter construct, 25ng pGL4.74 [hRluc/TK] vector and 500ng of an SV-driven SREBP-1 expression vector. Luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. The first intron was found to contain the cis-acting elements responsible for Ucp3 repression by SREBP-1. (4673)

Notes: U2OS and H1299 cells were transfected with expression vectors and analyzed for either protein levels or mRNA levels. Cells for mRNA level detection were transfected with the ViaFect™ Transfection Reagent (details not provided). Total RNA from siRNA treated cells was isolated and used in semi-quantitative RT-PCR with the PCR portion provided by the GoTaq® Hot Start Green Master Mix. The proteins ARID3A and ARID3B were expressed in with the TNT® Coupled Reticulocyte Lysate System for use in electrophoretic mobility shift assays. (4677)

Notes: The authors demonstrated that CellTiter-Glo® Cell Viability Assay could be used for anthelminthic drug discovery.  The pilot study identified hits previously reported to have some anti-parasite activity.  To demonstrate that the CellTiter-Glo® Reagent was able to penetrate the schistosomula, the reagent was mixed with the CellTox™ Green Cytotoxicity Assay and visualized by fluorescent confocal microscopy.  (4648)

Notes: The researchers in this study used Wizard^® Genomic DNA Purification Kit to extract DNA to be used in the next-generation sequencign of the I-Scel breakpoint in U2OS EJ-DR cells. (4891)

Notes: A robust ELISA-based histone deacetylase (HDAC) activity assay was developed for use with mammalian cells. The assay was then shown effective in screening selective HDAC inhibitors. Mammalian cell-derived HDAC isoforms, instead of recombinant proteins, was a key feature of this ELISA, developed using the HDAC-Glo™ Class IIa Assay (Cat.# G9560) and HDAC-Glo™ 2 Assay (Cat.# G9590). (4634)

Notes: These authors studied the interactions between G-protein coupled receptors (GPCRs)  and 13 different G-proteins using a NanoBRET assay. NanoBRET assays were performed using protein partners labeled with NanoLuc luciferase or Venus yellow fluorescent protein. The improvement in signal-to-noise ratio achieved using the NanoBRET method enabled resolution of signals from previously intractable G-proteins and GPCRs. The authors demonstrated that GPCRs engage multiple G-proteins with distinct patterns of activity or “fingerprints”. This differential engagement of multiple target G-proteins was revealed by quantitative analysis of G-protein activation kinetics.  (4588)

Notes: To track the fate of a microbe after it was consumed by a mosquito and migrated to the insect’s gut, a stably expressed, sensitive reporter gene that could track the bacteria with minimal effects from environmental factors was needed. The commensal bacteria Elizabethkingia anophelis was conjugated with an Escherichia coli strain carrying a plasmid with the NanoLuc® luciferase gene. Expression of NanoLuc® luciferase was measured by lysing the bacteria with Passive Lysis Buffer with lysozyme, mixing the resulting lysate with an equal volume of NanoGlo® Luciferase Assay Reagent and measuring luminescence. Larval Anopheles stephensi, Aedes triseriatus and Anopheles gambiae mosquito strains were incubated with NanoLuc® luciferase-expressing E. anophelis for 2 hours, samples taken at 0, 1, 1.5, 2 and 2.5 hours. Luciferase expression was determined by homogenizing four larvae, centrifuging the sample, washed with PBS then suspended in PBS, mixed with an equal volume of NanoGlo® Luciferase Assay Reagent and measured luminescence. Larval A. stephensi, A. triseriatus and A. gambiae mosquitos were fed NanoLuc® luciferase-carrying E. anopheles and reared to adult stage where mosquitos were homogenized and NanoLuc® luciferase expression measured. Adult A. stephensi were fed NanoLuc® luciferase-expressing E. anopheles in a 10% sucrose solution for 16–24 hours. After the 10% sucrose solution was replaced, samples of mosquitos were taken for several days and tested for NanoLuc® luciferase expression. (4573)

Notes: Primary normal human bronchial epithelial (NHBE) cells were cultivated on semi-permeable membranes of cell culture inserts exposed directly to vapor from 200 puffs of two different e-cigarette liquids (0% and 2.4% nicotine). Twenty-four hours post exposure, two assays were multiplexed to assess oxidative stress (ROS-Glo™ H₂O₂ Assay) and cell viability (CellTiter-Blue® Cell Viability Assay). First, the cells were incubated with 200µl of medium + 50µl of H₂O₂ Substrate Solution for 3 hours and 75µl of the mixture transferred to a white 96-well plate. An equal volume of Detection Solution was added, incubated for 20 minutes at room temperature and luminescence detected. The exposed cells had the remaining medium + H₂O₂ Substrate Solution removed and replaced with 300µl of fresh medium + 60µl of CellTiter-Blue® Reagent. After a 2-hour incubation, 100µl of solution was transferred to a black 96-well plate and fluorescence measured at 544nm_Ex/590nm_Em. (4569)

Notes: The authors were interested in developing a high-throughput screening method for detecting G_i-coupled receptors agonists but without the need for a cAMP inducer. HEK 293 cells were stably transfected using either pGloSensor™-22F cAMP Plasmid or pGloSensor™-22F cAMP Plasmid and a plasmid carrying an internal ribosome entry site (IRES) and succinate receptor 1 (SUCNR1) with puromycin resistance. HEK 293 cells stably carrying the pGloSensor™ plasmids were starved for 5 hours, detached from a T75 flask and incubated in the dark at room temperature with HBSS assay buffer containing 300μM IBMX and GloSensor™ cAMP Reagent. Cells were dispensed at 150,000 cells per well in 96-well plates or 37,000 cells per well in 384-well plates with various concentrations of SUCNR1 ligands. After 10 minutes, a basal level of luminescence was measured before forskolin or isoproterenol was injected (40 readings taken following injection). In some experiments, the cells were incubated 100ng/ml pertussis toxin the day prior to adding ligands or agonists. The assay system was then tested to measure the EC₅₀ value for succinic acid and compare to published literature. (4576)

Notes: The authors were interested in creating a genetic manipulation system to screen drugs for inhibiting or killing Cryptosporidium parvum. Sprorozoities were excysted from Cryptosporidium oocysts purified from infected calf feces that passed through an environment that mimicked stomach and intestinal passage. These sporozoites were then transfected using electroporation with plasmids carrying reporter genes including NanoLuc® luciferase flanked by C. parvum 5´ and 3´′ regulatory sequences from highly expressed genes. Then the transfected sporozoites infected human ileocaecal adenocarcinoma cells (HCT-8) and reporter activity measured after 48 hours. Of the reporter genes tested, only NanoLuc® Luciferase (Nluc) was able to be detected. Nluc was fused to neomycin and to create a Nluc repair assay, a termination mutant Dead Nluc was created. Sporozoites were transfected with either Dead Nluc alone or Dead Nluc plus Cas9 and specific guide RNA (gDNA), and luciferase activity measured. The Cas9 with gDNA restored Nluc activity. To further test this system and see if it could disrupt a gene associated with drug resistance, Nluc-Neo with Cas9 and gDNA targeted thymidine kinase. With the target gene removed, the parasite was more susceptible to antifolate trimethoprim. (4571)

Notes: Numerous cell lines were cultured for 72 hours in medium with glutamine, without glutamine or without glucose.  In all cell lines, glucose withdrawal was cytotoxic during the incubation.  The cells were highly tolerant of glutamine withdrawal with minimal cytotoxicity.  Cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay added at plating to cells in 12 well plates.  The fluorescence was measured every hour while incubated in an Essen BioScience IncuCyte FLR instrument. All cell lines used in the study were authenticated through STR analysis with the GenePrint® 10 System.  (4646)

Notes: Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles. aCell permeable phosphorescent nanoparticle probes designed to enable the study of cell and tissue oxygenation were assessed for cytotoxicity in monolayer culture and rat brain slices.  The probe demonstrated no significant cytotoxicity on cultured PC12, mouse endothelial fibroblasts, primary cortical or cerebellar granule neurons in concentrations and exposure time tested as judged with the CellTiter-Glo® Cell Viability Assay.  Rat brain slices (400µm) were incubated with and without the nano probes and cytotoxicity visualized by fluorescent microscopy following 3 hour incubation with 0.05% CellTox™ Green Dye.  The probe had no effect on cell viability in the 3 to 24 hour time frame.  (4645)

Notes: PC12 cells were plated and deprived of oxygen and glucose for 5-7 hours. Parallel samples were assessed for viability with the CellTiter-Glo® Cell Viability Assay and cytotoxicity with CellTox™ Green Cytotoxicity Assay (end-point method).  Deprived cells were also analyzed by RT-PCR following RNA extraction with the SV Total RNA Isolation System and converted to cDNA with the ImProm-II™ Reverse Transcriptase in the presence of RNasin® Ribonuclease Inhibitor and PCR performed with the PCR Master Mix for a limited number of cycles.  (4647)

Notes: A combination therapy of the chemotherapeutic cisplatin and an IL-1R antagonist was tested as a potential treatment in malignant mesothelioma (MM). The authors observed both caspase-1 activation and pyroptosis upon treatment. Caspase activation was measured using the Caspase-Glo® 1 assay, and pyroptosis was measured with the CellTiter® 96 Aqueous One Solution Assay. (5148)

Notes: The effect of tryptophan on skin hTERT immortalized normal keratinocytes was assessed at 24 and 48 hours with the RealTime-Glo™ Cell Viability Assay.  After the read at 48 hours, CellTox™ Green Cytotoxicity Assay was added to each well to measure dead cells per well using the end-point protocol. Tryptophan, which was determined toxic to Pseudomonas biofilms, was not cytotoxic to the human keratinocytes. (4644)

Notes: Induced pluripotent stem cells (iPSC) were differentiated into neurons and treated with 20, 100 and 500μM of ketamine for 6 and 24 hours to examine the neurotoxic effects of ketamine. The ApoTox-Glo™ Triplex Assay was used to examine cell viability and caspase-3/7 activation in the ketamine-treated iPSC-derived neurons. Fluorescence (cell viability) and luminescence (caspase-3/7 assay) was detected using a GloMax® multimode instrument. To examine if reactive oxygen species levels changed when iPSC-derived neurons were exposed to ketamine, the authors used the ROS-Glo™ H₂O₂ Assay with cells that were ketamine treated with or without a ROS scavenger for 6 and 24 hours. Luminescence was detected using a GloMax® instrument. Caspase activity was confirmed with or without the ROS scavenger using the Caspase-Glo® 3/7 Reagent from the ApoTox-Glo™ Triplex Assay. The levels of ketamine-induced oxidative stress were assessed in iPSC-derived neurons using the NAD/NADH-Glo™ Assay, and cellular ATP levels determined using the Mitochondrial ToxGlo™ Assay. The luminescence from both assays were measured on a GloMax® detection instrument. (4570)

Notes: The authors synthesized shell liposome–silica hybrid (LSH) nanoparticle (NP) made of a silica core surrounded by a multicomponent cationic lipid bilayer with and without polyethyleneglycol (PEG) grafted onto the lipid surface. They found that the negatively charged poly(I:C)-loaded LSH NPs are more efficient than their liposome counterpart in eliminating cancer cells. Cell viability of the test cells was measured using an MTT assay, and absorbance was measured using the GloMax® Discover. (4763)

Notes: SR-786 NPM/ALK-positive human anaplastic large cell lymphoma cell line was treated with lobatin B, a plant-derived natural compound. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and reversed transcribed into cDNA with the GoScript™ Reverse Transcription System. Levels of NPM/ALK and NF-κB expression was measured with the dye-based GoTaq® qPCR System. SR-786 cells were also analyzed for functioning metabolism with the CellTiter-Blue® Cell Viability Assay and apoptosis with the Apo-ONE™ Homogeneous Caspase-3/7 Assay. (4604)

Notes: Low-temperature atmospheric pressure plasmas (LTP) were examined for the mechanism of toxicity on cultured immortalized and primary prostate cancer cells. LTP induces an increase in H₂O₂ in the culture media as judged by the ROS-Glo™ H₂O₂ Assay leading to cell death as measured with the CellTox™ Green Cytotoxicity Assay. CellTox™ Green was quantified with a plate reader and verified by fluorescent microscopy. The cells experienced no caspase-3/7 activation as judged through use of the Caspase-Glo® 3/7 Assay, leading to the conclusion of necrotic cell death. (4711)