Notes: Primary hepatocytes from Sprauge-Dawley rats were incubated with various concentrations of tert-butylhydroperoxide to induce oxidative stress and then cell viability, cytotoxicity and apoptosis were assayed with Promega's CellTiter 96 AQueous One Solution Cell Proliferation Assay, CellTiter-Blue Cell Viability Assay, CellTiter-Glo Luminescent Cell Viability Assay, CytoTox-ONE Homogeneous Membrane Integrity Assay, Apo-ONE Homogeneous Caspase-3/7 Assay and assay systems. (2969)

Notes: Researchers used Promega's pGL3-Basic Vector to create a NF-κB promoter-driven firefly luciferase reporter vector for use in transient transfections with the phRL-TK Vector. The two vectors, along with pUC18, were transiently transfected into HEK293 cells using GeneJuice Transfection Reagent (Novagen). Transfections were performed overnight using 80ng NF-κB-pGL3-Basic construct, and 20ng of phRL-TK and pUC18 vectors in 96-well plates containing 2.5 x 10⁴ cells/well. The following day, the cells were exposed to various treatments. Luciferase activity was measured using the Dual-Glo™ Luciferase Assay System. (2672)

Notes: ImProm-II™ Reverse Transcriptase was used in real time RT-PCR to measure the ratio of Bax to Bcl-2 in immortalized rat proximal tubular cells (IRPTCs) cultured under normoxic or hypoxic conditions. The researchers used 1μg of total RNA in the reverse transcription reaction. Qiagen’s QuantiTest CYBR Green PCT Kit was used to quantify PCR products. Promega’s terminal deoxynucleotidyl transferase (TdT) was also used for TdT-mediated dUTP nick end labeling (TUNEL) assays on the cells. The TUNEL-stained cells were analyzed by FACS analysis. Data from these experiments was expressed as percent apoptotic cells.  (2849)

Notes: The Apo-ONE^® Homogeneous Caspase-3/7 Assay was used to assess caspase activity in human pulmonary artery endothelial cells (HPAECs) treated with varying amounts of recombinant angiopoietin-4 (Ang-4) with or without the Angiopoietin-1 inhibitor, Tie-2Fc.  Results were expressed as a percent of a control.  Examination of DNA fragmentation in apoptotic nuclei in cells treated under similar conditions confirmed the results.    (2782)

Notes: The pGL3-basic and pGL3-Promoter vectors were used to make constructs with varying lengths of the mouse LAMA1 promoter. Gene fragments up to 6.1 kb were cloned into the pGL3-promoter vector and analyzed for luciferase expression in a variety of cell lines using the Dual-Luciferase^® Reporter Assay System. F9, NIH/3T3, PYS-2 and EHS cells were transiently transfected with 200ng of reporter construct and 20ng of the Renilla luciferase-expressing phRL-null vector in 24-well plates.  Forty-eight hours later, cell lysates were harvested with Passive Lysis Buffer. Site-directed mutagenesis of  the LAMA1 gene promoter was performed with the GeneEditor™ in vitro Site-Directed Mutagenesis System.  (2631)

Notes: Cell chemotaxis was assessed by the CellTiter-Glo^® assay through rounds of directed selection. Data was presented as the migrating cells as a percent of total cells. (2913)

Notes: The Wizard^® SV Genomic DNA Purification System was used to purify DNA from cultured mammospheres. The mammosphere genomic DNA samples were then used in PCR reactions to amplify Short Tandem Repeats for genetic analysis. (2683)

Notes: The swarming behavior of the symbiotic-pathogenic bacterium Xenorhabdus nematophila is examined in this work. To investigate changes in gene expression in different strains at various times, the researchers utilized the AccessQuick™ RT-PCR System to measure the relative amount of gene product. To ensure that the assay was linear, different cycle numbers were used for each gene target ranging from 15 to 21 cycles of amplification. The RT-PCR used approximately 600ng of total RNA that was treated with RQ1 RNase-Free DNase before use.  (2745)

Notes: GAL4-recombinant desmoplakin (DP) domain constructs were immunoprecipitated from yeast extracts. Some immunoprecipitates were treated with Promega’s Calf Alkaline Phosphatase (CIAP). The immunoprecipitates were then analyzed by Western blotting with an anti-GAL4 antibody.  A change in the mobility of Gal4-DP was observed after CIAP treatment. (3263)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to measure cell death in mixed glia and microglia isolated from IL-18 or IL-1ß knockout mice. The cells were treated vehicle, IL-18, IL-1β or LPS for twenty-four hours before measurements were taken. (3139)

Notes: In this study, the authors investigated the interaction between the orphan receptors Dax-1 and Ad4bp/SF-1. Specific interaction was detected when the two proteins were used in both yeast and mammalian two-hybrid systems. To further characterize this interaction, an in vitro pull-down assay was performed. Dax-1 protein or a dax-1 mutant lacking an LXXLL motif were transcribed and translated in vitro using the TNT^® T7 Quick System and Fluorotect™ Green_Lys BODIPY labeled tRNA.  The fluorescently labeled protein interacted with a Ad4BP/SF-1-maltose binding protein (MBP) fusion protein immobilized to an amylose resin.  After elution and SDS-PAGE, the fluorescently labeled protein was detected using a Hitachi FMBIO^® II Fluorescence Imaging System. The interaction between Dax-1 and Ad4BP/SF-1 was dependent on the LXXLL motif. (3237)

Notes: In this paper, the role of microtubules on regulation of hypoxia-inducible factor (HIF) 1-α was explored. A549 human lung cancer cells were transiently transfected with 5µg of NFκB super-repressor plasmid or 3µg of either HA-tagged wild type or mutated HIF-1α construct using FuGENE® 6 Transfection Reagent in 6cm dishes. Twenty-four hours post-transfection, the cells were treated with vinblastine or colchicine before lysing the cells and analyzing the lysate by Western blotting using anti-HIF-1α or anti-HA antibodies (Figures 3 and 6 in article). A549, hepa1c1c7 and hepa1c4 cells were transiently cotransfected with 0.4µg of an iNOS luciferase construct or an NFκB-dependent luciferase vector and 4ng of a CMV Renilla luciferase plasmid in six-well plates. After six hours, the cells were treated with vinblastine, colchicine, nocodazole and paclitaxel and incubated for an additional 6–10 hours. The luciferase activity was then assessed using the Dual-Luciferase® Reporter Assay System (Figures 2 and 4). (4293)

Notes: The pGEM^®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

Notes: Nuclear receptor ligand binding regions from the Farnesoid-X-activated receptor (FXR) and peroxisome proliferator-activated receptor delta (PPARδ) were fused to a Gal4 DNA Binding Domain. These constructs were used to screen compounds from a LOPAC library of 640 compounds. Ligands bound to the Nuclear receptor ligand binding regions would result in the fusion proteins binding and inducing expression of a luciferase reporter construct with 5 upstream Gal4 binding element repeat sequences. For these experiments, Human hepatoma cell line (Huh7) cells transiently transfected with both plasmids were seeded in 384 well plates. After exposure to the various compounds in the LOPAC library luciferase expression was assessed with the Steady-Glo^® Luciferase Assay System. Cells and Steady-Glo^® reagent were added to cultures by a Labsystem Multidrop-384 liquid dispenser. Plates were read on a Molecular Devices ChemiLuminescence Imaging Plate Reader. (3265)

Notes: NF-E2-related factor-2 (NRF2) upregulates transcription of antioxidant response element (ARE)-driven genes. The authors examined the sensitivity of Nrf-2^–/^– primary mouse neurons to oxidative stress-induced apoptosis using the mitochondrial toxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPP⁺) and rotenone. Primary cultures were >090% neurons, as judged by immunostaining with the Anti-βIII Tubulin mAb and other neuron-specific antibodies. Neuronal cytotoxicity in the presence of MPP⁺ and rotenone was monitored using the CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay and TUNEL assays. The level of expression of the antioxidant-responsive gene quinone oxidoreductase was measured by mRNA quantitation using RT-PCR, enzyme activity assays and immunocytochemistry. The Reverse Transcription System was used to synthesize cDNA for subsequent amplification by PCR. (3570)

Notes: The CellTiter-Glo^® Luminescent Cell Viability Assay was used to assess the cytotoxic effects of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) on HeLa cells. Cells were assayed after a 3 hour treatment with 50μM MNNG. Data is presented as the relative light units observed with each sample. (2755)

Notes: pGEM^®-T Easy Vector was used to clone PCR products.  The CytoTox 96^® Non-Radioactive Cytotoxicity Assay  was used to determine specific cytotoxicity of human dendritic cells that were transduced with recombinant adenoviruses containing the gene encoding a fusion protein of granulocyte-macrophage colony stimulating factor and carbonic anhydrase IX. (2674)

Notes: Promega T4 Polynucleotide Kinase was used to end-label an oligo representing a NF-kB binding sequence element with [γ-³²P] ATP (7,000 Ci/mmol).  Specific primers to IFN-β and IκB-α messenger RNA were used in RT-PCR to generate products for cloning into the pGEM^®-T Easy Vector. The resulting plasmids were linearized with Nco I and Spe I, respectively, and used as templates for in vitro transcription using the Riboprobe^® System to generate probes for use in an RNase protection assay.  The antisense probes were labeled with [α-³²P]CTP (800 Ci/mmol) in the Riboprobe^® reactions.  RNase ONE™ Ribonuclease was also used in this study.  (3197)

Notes: Researchers used the Apo-ONE^® Homogeneous Caspase-3/7 Assay on rat kidney homogenates from normoxia (20% O₂) and hypoxia (8% O₂) treated rats. The authors homogenized rat kidneys in a well described buffer before clearing the homogenates by centrifugation at 50,000 x g and using them in the Apo-ONE^® Homogeneous Caspase-3/7 Assay. A Genius SpectraFluorplus fluorescence spectrometer with a 521nm filter was used to measure the relative fluorescence units obtained from the kidney lysates. (2698)

Notes: The CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay  was used to evaluate the effect of oxytocin on proliferation of human endometrial  endometriod adenocarcinoma cell lines. (2654)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cell-mediated cytotoxicity when a tumor cell line treated with Fas-neutralizing antibody was exposed to tumor-infiltrating lymphocytes. (2917)

Notes: These authors showed that double-substitution mutation of two N-terminal juxtaposed residues (from positive to neutral charged species) resulted in a reversal of the transmembrane orientation of the protein of interest. For in vitro transcription/translation, they used the TNT^® T7 Coupled Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes. A protease protection assay and an epitope-specific pull-down assay were used to determine the membrane orientation of the in vitro synthesized protein. (3051)

Notes: Researchers used PCR primers with Kpn I and Hind III restriction sites to clone human IL-2 from a known rhIL-2 E. coli clone containing the PTCGF-11 vector. The PCR product was digested with Kpn I and Hind III and cloned into the PinPoint™ Xa-3 Vector. Transformed E. coli JM109 clones were then pre-incubated in the presence of 8μM biotin for 2 hours before being induced with 100μM IPTG for an additional 2 hours. After induction, the cells were collected and resuspended in a lysis buffer before mechanical lysis with a French press. The lysate was then passed over a SoftLink™ Soft Release Avidin Resin column and the biotinylated rhIL-2 eluted. The resultant purified biotinylated rhIL-2 displayed similar properties and biological activity to native IL-2. Elutants from cells transformed with the PinPoint™ Xa Control Vector produced no biotinylated rhIL-2 and did not display any properties indicating that IL-2 was present. (2680)

Notes: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

Notes: To help determine the usefulness of a recombinant Adenovirus for gene delivery and vaccination, the authors compared various methods to determine the amount of anti-Adenovirus serotype 5 (Ad5) neutralizing activity present in human sera. This study describes a system using firefly luciferase in Ad5 vector to infect A549 human lung carcinoma cells.  Using a range of 8,000 to 5 virus particles/cell, the Ad5-luciferase was added to 10⁴ cells/well in a 96-well plate in a total volume of 200µl. After 24 hours, the medium was discarded, and 100µl of PBS was added to each well followed by 100µl Steady-Glo^® Luciferase Assay Reagent. After 15 minutes incubation at room temperature, 100µl from each well was transferred to a black and white isoplate and luminescence was measured on a 1450 Microbeta Trilux. An alternate neutralization assay method used the MTT-based CellTiter 96^® Non-Radioactive Cell Proliferation Assay to score the Ad-mediated cytopathic effect by staining for viable cells and subsequent analysis of optical density. (3095)