Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cell necrosis after treated SMCs with decorin to induce platelet-derived growth factor receptor. (2986)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify Bacillus anthracis chromosomal DNA, which was then used in PCR to amplify a luxN ortholog (named open reading frame BA5047). Amplified DNA containing the luxN ortholog region was then cloned into the pGEM^®-T Easy Vector. This construct, when transformed into Vibrio harveyi (AI-1^-, and AI-2⁺), allowed the detection of an upregulated signaling system by a bioluminescent assay. (3092)

Notes: These authors created a construct encoding a Renilla and firefly luciferase fusion protein to examine the efficiency of site-specific DNA repair. The Renilla gene was taken from the pRL-null Vector and the firefly luciferase gene originated from the pSP-luc+NF Fusion Vector. The fusion construct was created by ligating the C-terminus of the Renilla luciferase gene to the N-terminus of the firefly gene.  The fused proteins were expressed at a constant ratio when transfected into mammalian cells.  Firefly luciferase expression was eliminated by deleting a T at position 213 creating an ochre translational stop codon and placing the downstream sequence out of frame. The mutant protein was then tested for repair using two methods: small fragment homologous replacement and oligonucleotide-mediated repair. The Renilla:firefly expression ratio was tested in several human, murine and simian cell lines and assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System.  In addition, the repaired plasmids were recovered to verify the sequence correction. (3064)

Notes: The M3 gene product of gammaherpesvirus 68 (MHV-68) has been shown to bind certain chemokines and to block some chemokine signaling in vitro. This study demonstrates that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21. Approximately 30,000 Ba/F3-hCCR7 cells were used in a chemotaxis assay. After a 2-hour incubation at 37° in a 5% CO₂ atmosphere, cell migration was quantitated using the CellTiter-Glo™ Luminescent Cell Viability Assay. The number of migrating cells was determined via interpolation from standard curves.  (2702)

Notes: In this paper, the authors describe use of ImProm-II™ Reverse Transcriptase to transcribe cDNAs for Quantitative RT-PCR. Reverse transcription (RT) reactions were performed with 1μg total RNA from treated rat PC12 cells. Light Cycler reactions were setup with 1-3μl cDNA from the RT reactions. The researchers incubated cells with EGF and bFGF and analyzed HIF-2α mRNA levels. For these experiments, PC12 cells were incubated with 30ng/ml EGF or 50μM bFGF for 8 hours. Erk1&2 phosphorylation was examined by Western blot using the Anti-ACTIVE® MAPK pAb. Western results were visualized by chemiluminesent detection and analyzed with a digital luminescent image analyzer. The Dual-Luciferase® Reporter Assay System was used to analyze PC12 cell-expressed luciferase from pEpoEm1-luc and pEpoE-luc promoter constructs that were normalized with the pRL-TK vector. Cells were harvested 17-18 hours post transfection and treatment. (2725)

Notes: The TNT^® T7 Coupled Reticulocyte Lysate System was used to express precursors and truncated precursors of cowpea and Arabidopsis phosphoribosyl aminoimidazole synthetase (AIRS) in the presence of [³⁵S]methionine. Purified chloroplasts or mitochondria were added to the TNT^® T7 Coupled Reticulocyte Lysate in vitro transcription/translation reactions to monitor uptake of the protein into each organelle. The researchers also performed negative controls on the chloroplast or mitochondria uptake assays by adding 1 unit of apyrase or 20μm valinomycin, respectively.  (2688)

Notes: The effects of bromodichloromethane (BDCM), a byproduct of water disinfection processes, on trophoblast cells isolated from term human placentas were investigated. The CytoTox-ONE™ Homogeneous Membrane Integrity Assay was used to assess cell membrane integrity and LDH release after trophoblasts were exposed to BDCM for 24 hours. Data was expressed in arbitrary fluorescence units. Ultimately, little difference was observed between BDCM-treated and untreated trophoblasts. (2756)

Notes: The in vivo effect of the water-soluble polysaccharide fraction (CAWS) produced by Candida albicans was studied.  It is found that at high doses CAWS inhibited proliferation of spleenocytes and inhibited growth rate of cultured monophages (RAW264.7) in a dose dependent manner.  To test cell viability of RAW264.7, cells were plated RPMI1640 with gentmycin sulfate and 10% FCS to 5x10⁴ or 1x10⁵ cells/mL, allowed to attach to the dish for two hours, then incubated for 24 hours at 37° with 5% CO₂ in the presence of CAWS or sonifilan.  The cells were then counted or assayed for viability using CellTiter-Glo™. (2711)

Notes: The CytoTox-ONE™ Homogeneous Membrane Integrity Assay was used to assess the cytotoxic effects of tranexamic acid (t-AMCHA) on neural human normal progenitor (NHNP) or normal human dermal fibroblasts (NHDF) cells. Varying concentrations of t-AMCHA (0.0 mM, 31.25 mM, 150 mM, 300 mM, and 450 mM) were added to 70-80% confluent cells, which were then cultured for 8 hours. t-AMCHA showed no toxic effect on either cell line.  (3031)

Notes: Mouse hepatocytes (AML-12 cell line) were assayed for cytotoxicity to 1 mmol/L hydrogen peroxide with and without 20ng/mL IL-13 using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. For these assays 1 x 10⁵ cells/well were seeded in 96-well microplates and cultured for 24 hours before IL-13 treatment. One hour later, hydrogen peroxide was added to the cultures. The cells were then cultured for an additional 24 hours before analysis with the CytoTox-ONE™ Homogeneous Membrane Integrity Assay.  (3030)

Notes: The generation times of the EPC, HUVEC, and HMVEC cells were determined over a 4-day period. Cells were plated in a 96-well plate format at 2000 or 3000 cells/well in triplicate and were assayed daily using the CellTiter-Glo® Luminescent Cell Viability Assay. (3143)

Notes: The Wheat Germ Extract was used to demonstrate that the 3’ end of a viral mRNA acts as a tRNA-like structure (TLS). The tRNA-like structure was shown to accept a [H³]valine residue in the Wheat Germ Extract. The turnip yellow mosaic virus (TYMV) TLS structure was also shown to be able to function with ribosomes by donating [H³]valine in translation assays using Wheat Germ Extract with TYMV RNA but not BMV or AMV RNA. RNAs to be used as templates in the reactions were transcribed with the T7 RiboMAX™ Large Scale RNA Production System. After transcription DNA templates were removed with RQ1 RNase-Free DNase. (3386)

Notes: The Apo-ONE^® Homogeneous Caspase-3/7 Assay was used to demonstrate that 10U/ml of erythropoietin (EPO) can decrease apoptosis in primary cultured neonatal rat ventricular myocytes (NRVMs) under hypoxic conditions. The authors also describe using 1μM staurosporine and Ac-DEVD-CHO, at 5μl/well, as positive and negative controls, respectively. Cells were plated in 96-well tissue culture plates and treated in normoxic or hypoxic conditions for 24 hours before reading results using a Spectra Max GeminiXS fluorometer (Molecular Devices). (2839)

Notes: Plated at a density of 2500 cells/well in 96-well plates and allowed to recover overnight, the cells were then challenged for 1 hour with E-selectin monoclonal antibody or antibody drug conjugate, washed and allowed to proliferate in fresh growth media for four days. Cell viability was assessed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3152)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess the cytotoxic T-lymphocyte activity of patients suffering multiple-myeloma against autologous primary MM cells. (2971)

Notes: These authors characterized cytochrome P450 expression in mouse ovaries treated with the ovotoxin 4-vinylcyclohexene. Total mouse ovary RNA was reverse transcribed using the Reverse Transcription System and random primers, followed by quantitative PCR using primers specific for CYP2E1, 2A and 2B. (3442)

Notes: MCF-7 cells were transfected with a plasmid encoding constitutively active Akt-3. To assess estrogen-related transcriptional activity, the cells were transfected with an ERE-luciferase (firefly) reporter and a Renilla luciferase control plasmid. Dual luciferase assays were performed to determine the effect of estrogen on transcription in these Akt-3 expressing cells. Additionally, proliferation assays were performed using the CellTiter® AQ_ueous One Solution Cell Proliferation Assay and showed that the Akt-3 expression confers serum-independent growth on the cells. (2709)

Notes: Luciferase reporter gene constructs based on the pGL3 vector that contained either MMP-2 or MMP-9 promoters, SV40 promoter (positive control ) or the luciferase basic vectors were transfected into metastatic cells, S53JB-V cells or UMUC-3 cells along with a pβ-actin RL control construct. Cells were treated with an anti-IL-8 antibody, IgG control or no antibody. Dual luciferase reporter assays were performed to determine the effect of the treatments on activity and expression of the MMP-2 and MMP-9 constructs. (2716)

Notes: These authors analyzed gene expression profiles in the prostate cancer cell line PC3 upon induction of Akt activity to try to identify genes regulated by Akt that participate in the transformation of cells. They identified one mRNA of interest (Fra-1) and cloned its 5' regulatory region into a pGL3-Basic firefly luciferase reporter construct. This construct was used to transiently transfect MCF7 human breast cancer cells along with an Akt plasmid construct and a control vector expressing Renilla luciferase. The firefly construct was induced 4- to 5-fold by co-transfection with Akt3. Transfection conditions were as follows: MCF-7 cells were grown to 70% confluence in six-well plates, then incubated for 15 min with a mixture of 5ng of the control Renilla plasmid, 0.5μg of the Akt-expressing plasmid, and 0.5μg of pFra-luc construct and Fugene® 6 reagent at a 3:1 transfection reagent:DNA ratio. After 48 hours, luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System.

(4361)

Notes: These researchers used luciferase-containing T-DNA insertions in Arabidopsis thaliana for gene trapping. Luciferase was chosen because its transient expression allowed temporal expression studies. Several insertion vectors were constructed and found to have different insertion frequencies. Vectors containing the luc+ gene had substantially higher insertion rates than native luciferase vectors. Luciferase activity was measured in vivo with a CCD camera or, for longer term studies, with an automated scintillation counter sampling every 15-25 minutes over one week. The application of IRES sites in gene trapping experiments was also investigated using firefly and Renilla luciferases. The Dual-Luciferase® Reporter Assay System was used to monitor luciferase activity in vitro. Finally, to sequence the T-DNA insertion sites, genomic DNA was isolated from T₂ seedlings using the Wizard® Magnetic 96 DNA Plant System and was subsequently amplified and sequenced. (2787)

Notes: A human cDNA library of ~20,000 sequences was tested for putative modulators of the activator protein-1 (Ap-1) signal transduction pathway. The plasmid library was co-transfected with luciferase reporter plasmids containing AP-1, p53 or Epo response elements. Transfections were performed in 384-well plates using HEK293, HCT116 or HepG₂ cells. After 48 hours, the Bright-Glo™ Luciferase Assay was used to determine the level of luciferase activity in the transfections. Luminescence was read using an Acquest Plate Reader (LJL Biosystems) (3299)

Notes: Researchers performed a 5´ RACE analysis on the human CCR8 and mouse CX3CR1 transcripts.  PCR-amplified products from the reactions were cloned in to the pGEM^®-T Vector for further analysis.  Also, the human CCR8 and mouse CX3CR1 promoters were cloned into the pGL3-Basic Vector and transfected into THP1 and Jurkat cells. For transfections, 15 x 10⁶ cells in 750μl were used with 15μg of construct in electroporation reactions. Transfectants were grown for 48 hours and analyzed with the Bright-Glo™ Luciferase Assay System. A fourfold increase in activity was observed compared to the pGL3-Basic Vector alone. (2729)

Notes: Cultured MASMCs were serum starved and stimulated with platelet-derived growth factor, and after four days, the cells were assayed by the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3159)

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess the proliferation of both Jurkat cells and peripheral blood lymphocytes (PBLC) treated with various strains of Helicobacter pylori and isogenic Campylobacter jejuni or Escherichia coli HB101. In addition, the proliferation of PBLCs and Jurkat cells was assayed after treatment with concentrated bacterial culture supernatant with or without vacuolating cytotoxin VacA. (3144)

Notes: The pGL3-Control Vector was co-transfected into COS-7 cells with mammalian expression vectors expressing either heat shock protein Hsp70 or Hsp105α.  The cells were ATP-depleted in glucose-free media with 2μM rotenone and 5mM 2-deoxyglucose for up to 3 hours. Afterwards the cells were lysed with the Cell Culture Lysis Reagent and assayed for luciferase activity uaing the Luciferase Assay System. ATP depletion was verified during 2μM rotenone and 5mM 2-deoxyglucose treatment using the CellTiter-Glo^® Assay.  (2841)