Notes: Cow (Bos taurus), horse (Equus caballus), sheep (Ovis aries), antelope (Antilocapra americana), dog (Canis familiaris), cat (Felis catus), and rabbit (Oryctolagus cuniculus) genomic DNA were obtained by extraction from tissue and blood samples using the Wizard^® Genomic DNA Purification kit. SINEs (short interspersed elements) PCR was performed on various combinations of purified DNA from the above species. (3260)

Notes: The CellTiter-Glo^® Luminescent Cell Viability Assay was used to measure viability of human breast cancer MCF-7 and MCF-7.3.28 cells after 24 hour incubation with roscovitine (ROSC).   A dose-dependent cytotoxic effect was observed on 60-70% confluent cells with increasing ROSC concentrations from 0.01 to 100μM. Data are expressed as percent viable cells compared to untreated controls. (2840)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess antibody-dependent cell-mediated cytotoxicity using rhuCD40 mAb on B cells cocultured with effector mononuclear cells at various effector to target ratios. (2966)

Notes: This paper describes amplification of rat NHE1 cDNA using primers incorporating the Xho I and Xba I restriction endonuclease sites, Kozak consensus, ATG start site, and a stop codon. The resultant 1.1 kb NHE1 encoding-PCR product was digested with Xba I and Xho I and cloned into the pTNT™ Vector.  This construct was used as a template in a TNT^® T7 Quick Coupled Transcription/Translation reaction to generate substrates for an in vitro caspase-3 cleavage assay. Proteolytically cleaved fragments of NHE1 were run on a polyacrylamide gel and visualized by autoradiography. (3063)

Notes: The CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay was used to measure metabolic activity of germinating Glomus intraradices fungal spores over time. For this assay, 400 spores were germinated in cuvettes for three hours at 25°C before addition of the CellTiter96^® AQueous Non-Radioactive Cell Proliferation Reagent. Absorbance readings were taken on the cuvette every 15 minutes for 8 hours. (2685)

Notes: The pGL3-Promoter Vector was treated with various concentrations of the chemotherapeutic cancer and DNA cross-linking agent, cisplatin. The damaged pGL3-Promoter Vector and the pSV-β-Galactosidase Control Vector were then transiently transfected into the human lung adenocarcinoma cell line, A549, and later analyzed for luciferase activity using the Luciferase Assay System with Reporter Lysis Buffer. The researchers also used M-MLV Reverse Transcriptase to clone the antisense sequence to XPA (Xeroderma Pigmentosum group A) mRNA.  The cloned antisense sequence was then expressed in A549 cells for its effect on XPA gene expression. (2833)

Notes: The CellTiter 96^® AQueous One Solution Cell Proliferation Assay was used to analyze the effect of siRNA transfection on HeLa cell proliferation. HeLa cells were transfected twice with 10nM of a siRNA for RNA helicase II/Guα, a scrambled sequence siRNA, or mock transfected with the transfection reagent. Transfected cells (50,000-100,000) were seeded in 6-well culture plates for 72 hours. After this incubation, cells were plated at 625-20,000 cells per well in 96-well culture plates, and were then assayed using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay.  (3036)

Notes: The β-Galactosidase Assay  and Luciferase Assay were used to assess the effects of siRNAs on the expression of β-catenin in colon cancer cells. (2647)

Notes: The species-specific response of mouse hepatoma (Hepa.2D) and guinea pig adenocarcinoma (GPC.16) cells to flavonoid treatment was explored in this study. Cell lines stably transfected with dioxin responsive element-driven luciferase constructs were treated with a series of flavanoids. Luciferase expression was measured using the Steady-Glo®> Luciferase Assay system. To increase the number of cells available, cells were grown on Cytodex-1 beads in a larger volume, then split into an opaque 96-well plate prior to application of the Steady-Glo® Reagent. Total RNA was isolated using the SV Total RNA Isolation System, then used in real-time PCR to quantitate luciferase and GAPDH RNA levels after treatment. (3091)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from Tobacco Hornworn (M. Sexta) larvae. The RNA was reverse transcribed with primers specific to the δ-endotoxin-binding region from the cadherin ectodomain of BT-R1. (2785)

Notes: The role of calcineurin in linking calcium signaling to transcriptional activation was explored in this study. To do this, T-cell activation in calcipressin knockout mice was investigated.  Transcriptionally activated cells were generated by incubating 1 x 10⁵ CD4⁺ T-cells with plate-bound anti-CD3 or anti-CD28.  The viability of these cells was measured daily using the CellTiter-Glo^® Luminescent Cell Viability Assay. Activated cells were stained with either anti-Fas ligand and anti-CD4 antibodies or with anti-IFN-γ antibody and allophycocyanin-conjugated IL4, and analyzed by flow cytometry.  (2724)

Notes: In this paper, FuGENE® reagent was used to transiently transfect HUVEC 293 cells. Transfection conditions were as follows: HUVEC cells were plated at 1 X 10e5 cells/well in 12-well plates 24hr prior to transfection with 0.55µg DNA at a 4:1 FuGENE:DNA ratio. (4398)

Notes: The authors explored the role of beta-catenin and T-cell factor 4 to transactivate vimentin, a protein involved in gain of mesenchymal characteristics and loss of epithelial characteristics as a precursor to metastasis. The vimentin promoter was cloned into the pGL3-Basic Vector. The beta-catenin/TCF binding sites upstream of a minimal c-fos promoter drove firefly luciferase expression in plasmids called TOP-FLASH (wild-type binding sites) and FOP-FLASH (mutant binding sites).  To examine beta-catenin/TCF-4 induction, several human mammary epithelial cell lines were transfected with a mixture of 0.15µg of various firefly reporter constructs (wild type vimentin promoter, mutant vimentin promoter, TOP-FLASH,or FOP-FLASH), 0.15 µg of the beta-catenin expression vector (or the corresponding empty vector), 0.15 µg of the TCF-4 expression vector and 0.8 ng of the Renilla luciferase vector, phRG-TK.  Twenty-four hours after transfection, the cells were lysed and assayed using the Dual-Luciferase^® Reporter Assay System. (3087)

Notes: To investigate how interferon gamma stimulates expression of the murine Clara cell secretory protein (CCSP) gene, the 280bp CCSP promoter region was radiolabeled and incubated with nuclear extract from mouse transformed Clara cells (mtCC). Transcription factor binding sites were identified using the Core Footprinting System. A 30bp section of the CCSP promoter containing three transcription factor consensus sites was synthesized with 0, 1 or 2 mutated binding sites and tested in the presence of mtCC nuclear extract. Oligos and nuclear extract were allowed to form complexes with or without interferon gamma in Gel Shift Binding Buffer. A 166bp section of the CCSP promoter was also cloned into the pGL3-Basic Vector and co-transfected with the pRL-TK Vector into mtCC. The cells were subjected to interferon gamma treatment and the cell lysates assayed using the Dual-Luciferase^® Reporter Assay System. (3112)

Notes: Researchers created promoter constructs in the pGL3-Basic vector to study NHE3 promoter function in cotransfection experiments with the pRL-null vector.  Transfected Caco-2 cells were analyzed by the Dual-Luciferase^® Reporter Assay System. Beta-Galactosidase Assays were also performed on SL2 cells cotransfected with the pRL-null vector. The Renilla Luciferase Assay System was used to normalize these transfectants. (2642)

Notes: Researchers used luciferase reporter constructs along with pRLSV40 or phRL-null Vectors in transiently transfected NIH3T3, CHO, 293, HeLa, and Rat chondrosarcoma cell lines.  Transfections were performed in 6-well plates. Lysates of the transfectants were prepared at various times post-transfection and were analyzed with the Dual-Luciferase^® Reporter Assay System.  (2726)

Notes: The Y-box-binding protein YB-1 was examined through the use of a reporter plasmid with wildtype and mutant putative Y-box binding sites. The binding sites were constructed in the pGL3 Basic Vector and measured using the Luciferase Assay System. Studies were performed in normal human dermal fibroblasts.  Expression of YB-1 was found to repress expression of the COL1A2 gene at the transcriptional level. Confirmation of this dose-dependent inhibition was through measurement of the steady-state mRNA levels by quantitative, real-time RT-PCR.  The reverse transcription portion of the quantitative, real-time RT-PCR reactions were performed with ImProm-II™ Reverse Transcriptase followed by a TaqMan-type quantitative PCR step. (2623)

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess the viability of HepG2 cells stably expressing CYP3A4 response elements under low or no serum conditions (10-0% FBS). (3145)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess background cell death before the primary cells were used for too many passages. (2894)

Notes: Mig-6 cDNA was cloned into the pSI Vector to be used in cotransfection experiments with luciferase reporter constructs.  The phRL-CMV Vector was added to the transfections to normalize the data generated by the Dual-Luciferase^® Reporter Assay System in NIH/3T3 cells. (2632)

Notes: Immortalized mouse embroyonic fibroblasts (MEFs) from PARP-1 -/- and PARP-1 +/+ mice were exposed to a novel anticancer drug, named CHS 828. The cytotoxic effect of increasing concentrations of CHS 828 was assayed with the CellTiter-Glo® Luminescent Cell Viability Assay. For these experiments the researchers incubated the cells in CHS 828 containing media for 25 hours before assessment. (2860)

Notes: Promega's CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay was used to examine the effects of adenosine and adenosine receptor agonists on a pituitary folliculostellate cell line and two pituitary endocrine cell lines. The authors also used RQ1 RNase-free DNase during the course of their experiments. (2495)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess antiviral compound efficacy on cells infected with a GFP-expressing HCMV. Instead of assaying the media, the researches evaluated the residual cell layer. (2909)

Notes: In this paper, Promega Wheat Germ Extract System was used to express the oncogene product c-Abl and a mutant (Abl-PP). These in vitro translated proteins were assayed by Western blotting and immunoprecipitation. The Dual-Luciferase^® Reporter Assay System was also used during the course of these experiments. (2493)

Notes: In this study of plant steroid hormone (brassinosteroid) signaling pathways, the TNT^® T7 Coupled Reticulocyte Lysate System was used for in vitro transcription/translation of proteins that were then used in GST pull-down assays to study protein:protein interactions. (2429)