Notes: Purified GST-CNA-1 and GST-CNB-1 fusion proteins created from C. Elegans cna-1 and cnb-1 cDNA, two genes whose products resemble insect and mammalian calcineurin, were tested in the Serine/Threonine Phosphatase Assay System. Sixteen picomoles of each purified fusion protein were used per reaction. Data were expressed as pmol phosphate release/min/μg protein. The reactions were also incubated with 0.2mM EGTA or 1μM each of cyclosporin A and bovine cyclophilin to show specific inhibition of gene product activity. (2754)

Notes: Promega's Anti-Cytochrome C monoclonal antibody was used  in immunocytochemistry to detect mitochondrial release of Cytochrome C in apoptotic IMR90-E1A cells microinjected with caspase-2 expressing constructs. For this procedure, cells were fixed in 3% paraformaldehyde, washed, then permeabilized with 0.1% Triton X-100. Dilutions of Anti-Cytochrome C monoclonal antibody were applied, then visualized using Alexa Fluor 633 (Molecular Probes)-labeled secondary antibody, and a Leica TCS NT laser scan microscope equipped with a 488–534nm argon laser and a 633 nm He-Ne laser. The researchers also used the TNT^® T7 Coupled Reticulocyte Lysate System for in vitro transcription/translation of RAIDD/CRADD and caspase-2 deletion mutants. Templates for the transcription/translation reaction consisted of cDNA cloned into pCDNA₃-HA vectors.  The proteins of interest were labeled with ³⁵S-labeled amino acids and were used in protein association assays with GST-caspase-2 protein.  (2667)

Notes: Pam-212 keratinocytes (murine) or freshly isolated epidermal cells were treated in PBS with a variety of skin irritants in an attempt to stimulate release of ATP from the cells. Supernatants from the cell cultures were examined for ATP concentrations using a  luciferin-luciferase assay and  for LDH release using Promega's CytoTox 96^® Assay.  The percent -specific release of LDH was calculated  based on total LDH concentration detected in the supernatants after permeabilization with 0.3% Triton X-100. The authors conclude that injured keratinocytes release ATP. (2431)

Notes: The sterol regulatory element-binding protein-1a (SREBP1a) was in vitro transcribed and translated from a plasmid template using TNT^® T7 Quick for PCR DNA. A control reaction was performed using Transcend™ tRNA to confirm that the 120KDa protein was expressed correctly.  Unlabeled SREBP1a was used in gel-shift assays with a labeled oligo representing the SRE motif from the human ABCD2 gene promoter.  (3222)

Notes: The promoter region of human CYP19 was amplified from a lambda library and cloned into the pGEM^®-T Easy Vector for sequencing. Several mutants of this aromase cytochrome P450 promoter were created and cloned into the pGL3-Basic Vector. One microgram of the various reporter constructs along with 25ng pRL-null Vector (as an internal control) were transfected into HMEC-1 and MCF-7 cells in six-well plates. Luciferase levels were assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System. To further characterize the CYP19 promoter, a 30-mer region with a GATA motif was added to 250ng HMEC-1 nuclear extract in the presence of oligonucleotide competitors or antibodies using the Gel Shift Binding 5X Buffer, then analyzed by gel electrophoresis. (3110)

Notes: Neurons were seeded in polylysine-coated 24-well plates at a density of 4 x 10⁵ cells/well. After 48 hours, the cells were treated with varying concentrations of capsaicin (0-150 µM) in DMEM with or without 10% FBS for 18 hours before testing the viability with the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3149)

Notes: In this paper, cytotoxicity results obtained using an MTT-based assay were confirmed using the CellTiter-96^® AQ_ueous Non-Radioactive Cell Proliferation Assay and the CellTiter-Glo™ Luminescent Cell Viability Assay on primary human vaginal keratinocyte cultures and immortalized VK2/E6E7 cells. (2605)

Notes: In this paper, the SV Total RNA Isolation System was used to isolate total RNA from Gigaspora margarita. The authors reported isolating total RNA from 100 spores or 100mg of mycorrhizal roots. One microliter of the isolated RNA was used in RT-PCR to amplify Gigaspora margarita metallothionein (MT)-like polypeptide (GmarMT1) RNA. The researchers also cloned the GmarMT1 coding region using the pGEM^®-T Vector. (3077)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess inhibitors of a HCMV protein kinase on viral cytotoxicity. (2910)

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess the CTL activity of mouse splenocytes removed from mice immunized with a DNA vaccine and challenged with various H5N1 influenza virus strains. (2958)

Notes: Drosophila Mi-2 was discovered to specifically bind the DNA binding domain of dDREF (DNA replication-related element factor) by yeast two hybrid screen. This binding was shown to inhibit dDREF’s transcriptional regulatory action on a DNA replication-related element (DRE). Analysis of dDREF binding to the DRE was performed by examination of expression from –168DPCNAluc, –168mutΔPCNAluc, and pRL-actin5C reporter genes in Schneider cells using the Dual-Glo™ Luciferase Assay System. (2622)

Notes: The pAdVAntage™ Vector was cotransfected with a pCAT-control vector into 293 cells. A CAT assay was performed 48 hours post-transfection and results were compared to those from cells cotransfected with a pCAT-control vector and an experimental vector expressing influenza A virus NS1 protein.  In all transfections the total amount of DNA used was 2.5μg. (2753)

Notes: Total RNA was isolated from TOP10F’ or INVaF' E. coli (Invitrogen) using the SV Total RNA Isolation System. The isolated RNA was used in RT-PCR to detect cleavage of marA mRNA. A marA drug resistance gene sequence was also ligated into a chloramphenicol acetyltransferase (CAT) reporter vector. This construct was used to measure single-stranded nanovector-transcribed ribozyme activity on the marA sequence. CAT activity was measured using the CAT Enzyme Assay System. CAT activity was normalized by cotransfecting cells with the pSV-β-Galactosidase Vector and assaying lysates with the β-Galactosidase Enzyme Assay System. (2794)

Notes: The authors investigated the ability of histone deacetylase inhibitors to inhibit anchorage-dependent growth in non-small cell lung cancers. Anchorage-dependent growth was measured using the CellTiter 96^® Non-Radioactive Cell Proliferation Assay. (2440)

Notes: This study explores the utility of indirect cell viability measurements based on metabolic activity for monitoring neural stem/progenitor cells. The tetrazolium salt WTS-8 and the CellTiter-Glo^® Luminescent Cell Viability Assay were compared to BrdU incorporation for their ability to monitor cell proliferation. The authors conclude that both WTS-8 and CellTiter-Glo^® can accurately monitor cell proliferation, but that the increased sensitivity and ease-of-use of CellTiter-Glo^® assay made it an ideal choice for this application. (3123)

Notes: Hipppocampal neurons were cultured for 10-12 days and then hypoxic or ischemic conditions were induced. Eight hours after this event acute cell death was quantified by measurements of LDH release using Promega's CytoTox 96^® Non-Radioactive Cytotoxicity Assay. (2439)

Notes: BE(2)-M17 and HEK-293T cells (Human dopaminergic neuroblastoma and human embryonic kidney cells, respectively) were cultured in Opti-MEM (Life Technologies) with 10% FBS, penicillin (100 units/ml) and streptomycin (100 mg/ml). Cells were plated at 80% confluence and maintained in an atmosphere of 5% CO₂ at 37°C for twenty-four hours prior to transfection in 24-well culture plates. Firefly Luciferase-containing constructs (pGL3) were co-transfected with the phRL-TK synthetic Renilla luciferase vector, at a molar ratio of 1:100 (phRL-TK:pGL3).  These transfections were performed in serum-free media for 12 h using a 1:3 molar ratio of DNA: Fugene reagent (Roche Biochemicals), and 0.2 mg of DNA per well. After forty hours cells were rinsed with PBS and then harvested with Passive Lysis Buffer. The Dual-Luciferase^® Reporter Assay System was used to measure firefly and Renilla luciferase activities from the transfected cells using duplicate readings on a Turner Designs 20/20 Single-Injector Luminometer. (2663)

Notes: Researchers PCR amplified and cloned the Trl promoter into the pGEM^®-T vector. The Trl promoter sequence, as well as the eve promoter sequence and/or a CMV promoter, were used to make luciferase reporter constructs using the pGL3-Basic Vector. Transient transfection experiments were performed with the promoter constructs and GAGA factor expression constructs. Relative luciferase activity from each experiment was measured and compared to that of a control.  (2775)

Notes: Transiently transfected primary neuronal cells expressing beta-galactosidase were treated with NMDA and stained with X-Gal using the Promega method. Cytotoxicity of the treated cells was analyzed using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay. (2580)

Notes: In this paper, Promega's GeneEditor™ in vitro Site-Directed Mutagenesis System was used to change amino acids important for the nuclear localization signal and the kinase domain of the LBK1 tumor suppressor. Mutants were tested for their ability to arrest the cell cycle as well as for cellular localization. The pCI-neo Mammalian Expression Vector and the Dual Luciferase^® Reporter Assay System were also used during the course of this study. (2494)

Notes: In this paper, biotinylated and fluorescently labeled DNA probes were used simultaneously in hybridization reactions with restriction digested human genomic DNA. Streptavidin-paramagnetic particles were used to capture restriction fragments of interest prior to analysis for CpG methylation and short-nucleotide repeats with an alkaline-phosphatase-conjugated anti-fluorescein antibody.  These immuno-DNA complexes were then analyzed using the AttoPhos^® AP Fluorescent Substrate System in 96-well plates. Fluorescence signal was determined using a FluoroCount™ microplate reader. Details of  hybridization conditions, probe concentrations, washes and statistical analysis are provided. (2655)

Notes: Researchers stably transfected a vector containing a luciferase reporter gene under the control of Tp53 DNA binding site concatemers into Hs 766T human pancreatic cancer cells. The cells were then plated in 96-well plates and treated with a concentration of 2μg/ml of one of 16,000 individual compounds. Cells were then screened for up-regulation of Tp53-induced luciferase gene expression using the Steady-Glo^® Luciferase Assay System.  Results were analyzed by Wallac Trilux plate reading luminometer. Erratum in: Carcinogenesis. 2003 Oct;24(10):1713. (3195)

Notes: This study investigated the effect of aberrant expression of EAAT2 mRNA on expression of the EAAT2 protein in human glioma cells. EAAT2 encodes an excitatory amino acid transporter that helps to clear glutamate from synaptic cleft. In transfection-based studies on human U251 gliomal cells,  transient transfection efficiency was evaluated using a luciferase reporter gene, and luciferase activity was detected with the Luciferase Assay System. In separate experiments, RTPCR products were cloned into plasmid vectors. Plasmid DNAs containing PCR products were isolated from bacteria using the Wizard^® Plus SV Minipreps DNA Purification System. (2607)

Notes: An IL-1 inducible fragment of the murine IL-2 promoter and five tandemly-arranged NFκB binding sites were cloned into the pGL3-Basic Vector, creating the constructs pGL3-IL-2 and pGL3-5xNFκB, respectively. IL2 promoter activation and NFκB activation were measured after rhIL-1α - or PMA-stimulation of EL4D6/76 cells transiently transfected with these constructs. Luciferase activity was measured using the Steady-Glo® Luciferase Assay System. As an internal control, cells were co-transfected with the pRL-SV40 Vector, and expression of firefly and Renilla luciferase was measured using the Dual-Luciferase® Reporter Assay System. (2427)

Notes: The researchers used pGL3 basic constructs and the phRL-TK vector in cotransfection studies to examine the effect of hypoxia on promoter regions of DEC1 and DEC2 promoter constructs in ATDC5, 293T, and HeLa cells. The Dual-Luciferase^® Assay System was used to examine the expression levels of luciferase from the constructs. (2630)