Notes: In this study, the effect of arginosuccinate synthase (AS) on nitric oxide (NO) signaling was explored in bovine aortic endothelial cells. Expression of arginosuccinate synthase was knocked down using in-vitro-transcribed siRNAs. Using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay and the Apo-ONE® Homogeneous Caspase-3/7 Assay, it was found that reduction of AS leads to the induction of apoptosis. Detection was performed on a BMG FLUOstar spectrofluorometer. Supplying the cells with an NO donor decreased apoptosis induction. The regulatory role NO has on apoptosis is not known. (3065)

Notes: In this paper, the effect of a 5’ untranslated region from the Bcl-2 gene transcripts on firefly and Renilla reporter constructs was evaluated. A number of studies were performed using various single- and dual-reporter constructs containing the Bcl-2 5’ UTR. These constructs were transfected into 293T cells and assayed for luciferase activity using the Dual-Luciferase^® Reporter Assay System. Transfection studies with firefly luciferase mRNA constructs were also performed. In these experiments, firefly luciferase levels were measured using the Luciferase Assay System.  Transfections were normalized using the pSV-β-Galactosidase Control Vector and the Beta-Glo^® Assay System.  (3125)

Notes: Microsomal extracts from Jurkat cells were assayed for cytochrome P450 1B1 (CYP1B1) using the P450-Glo™ CYP1B1 Assay. CYP1B1 activity increased when Jurkat cell cultures were supplemented with 10nM biotin. (3132)

Notes: Aliquots of 1.8 x 10³ cells were placed in 96-well microtiter plates and the cells were allowed to adhere for 6 hours before medium containing two-fold serial dilutions of Aspergillus niger-derived antibody from 20 to 0.078 g/ml (final concentrations of 10 to 0.039 g/ml) or medium alone then were added to the wells. After 72 hours incubation, relative proliferation was measured by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (3153)

Notes: Primary T cells activated with anti-CD3 and anti-CD28 were treated with 2 µM of two calcium inhibitor compounds from a library of 16,000 plus cyclosporin A (CsA). The two compounds were able to reduce the apparent IC50 of CsA for the suppression of T cell activation without affecting the cell viability as measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3141)

Notes: The Renilla Luciferase Assay System was used to analyze mouse hepatitis coronavirus strain A59 (MHV-A59) entry in to cells. A mouse hepatitis coronavirus construct expressing Renilla luciferase was used to infect LR7 cells in the presence or absence of a Furin protease inhibitor. (3041)

Notes: Leaves of Glycine max (soybean) were co-transfected by particle bombardment with various combinations of vectors encoding plant disease resistance (R) genes and a luciferase reporter construct containing the constitutive ³⁵S promoter of Cauliflower mosaic virus. Leaf disks from the transfected areas were frozen in liquid nitrogen, ground, and resuspended in 240μl of Cell Culture Lysis Reagent. The lysates were then assayed for luciferase activity with the Luciferase Assay System. The luciferase values correlated to plant leaf cell survival of the various constructs. (3035)

Notes: Human U251 glioblastoma cell lines treated with antisense morpholino oligonucleotides were assessed for viability and apoptosis by multiplexing the CellTiter-Blue® Cell Viability and Apo-ONE® Homogeneous Caspase-3/7 Assays on single cell cultures. Cell viability was measured 4 hours after the addition of the CellTiter-Blue® Cell Viability Reagent to the cultures. Next, apoptosis measurements were performed on the same cell cultures by adding Apo-ONE® Homogeneous Caspase-3/7 Assay reagent to the cultures. Caspase-3/7 activity was then measured 12 hours later. (3168)

Notes: cDNA was synthesized from 5μg total RNA from patient skin fibroblasts using the specific ALG1 β1,4 mannosyltransferase primer in the presence of 5% DMSO and 1 unit of ImProm-II™ Reverse Transcriptase. Reverse transcription reactions were performed at 42°C for one hour.  PCR amplification of the cDNA produced ~1,200bp amplimers.  (3180)

Notes: Differences in the activation of apoptotic pathways by the TRAIL death receptors DR4 and DR5 were investigated. An siRNA screen was performed on HCT15 cells in 384-well plates to find genes that influence DR4- or DR5-mediated apoptosis differently. After induction of apoptosis using anti-DR4 or DR5 cross-linked antibodies, cell viability was assessed using the CellTiter-Glo® Reagent. It was found that the Signal Recognition Particle (SRP) plays a major role in DR4- but not DR5-mediated apoptosis. To further investigate this difference, stable HeLa cell lines were generated that expressed short hairpin RNA (shRNA) directed against SRP. The effects of various inducers of apoptosis were tested in these cell lines. Caspase activation was measured using the Caspase-Glo® 3/7 reagent. (3191)

Notes: In this paper, researchers examined the effect of activating mammalian homologues of the Drosophila Toll receptor gene (TDR) on apoptosis in the trophoblast cell lines HTR8 and 3A. Bacterial peptidoglycan (PDG) was used to indirectly activate caspase-3/7, -8 and -9 through TDRs. Caspase activity was measured using the Capase-Glo™ 3/7, 8 and 9 Assays. Caspase-3/7, -8 and -9 activities decreased in both the HTR8 and 3A cell lines transiently transfected with a Fas Associated Death Domain (FADD-DN). Data were expressed as relative light units and were normalized to protein levels in lysates. (3171)

Notes: A2L2 cells (1.5 × 10³ cells per well) were plated into 96-well flat-bottom plates. After 24 hours incubation at 37°C, drugs, vehicles and controls (medium and cells) were dispensed in duplicate into the appropriate wells.  The drugs tested were Doxorubicin, at concentrations ranging from 0.0625 to 125µg/ml, and paclitaxel, at concentrations ranging from 0.1875 to 375µg/ml.  The CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay was used to assess cell viability at 24 and 48 hours post-treatment. (3146)

Notes: To study the effect of the DNA intercalating chemotherapy agent doxorubicin, the viability of HCT116 cells plated 10⁵ cell/mL were tested over a titration of doxorubicin treatment for 48 hours. Using the CellTiter 96® Aqueous Cell Non-Radioactive Cell Viability Assay, the researchers found that increasing concentrations of the drug caused increasing cytotoxicity. However, testing for caspase activation with the Caspase-Glo™ 3/7 assay revealed that apoptosis was only induced in a narrow window of these concentrations. This helped elucidate the mechanism of apoptosis induction in doxorubicin treated cells. (3214)

Notes: This paper describes an assay to test BDNF dissociation from FluoSpheres (amine-modified microspheres, 1μM diameter; Molecular Probes). BDNF was covalently attached to the microspheres. Release of BDNF was tested in supernatants from the BDNF-microspheres in PBS, in complete media, and in culture with rat Dorsal root ganglia. Promega’s BDNF Emax® ImmunoAssay System was used to determine the amounts of BDNF in these samples. The DeadEnd™ Fluorometric TUNEL System was used to examine rat Dorsal root ganglia cells that had been transfected with either dynamitin or β-Galactosidase. In the TUNEL experiments, cells were fixed in 4% paraformaldehyde and counterstained with DAPI. Cells were also stained with Promega’s Anti-β-Galactosidase, Purified Monoclonal Antibody. For these immunocytochemical stains, a 1:500 dilution of Anti-β-Galactosidase antibody and a 1:1,500 dilution of a secondary antibody conjugated to Alexa Fluor-488 (Molecular Probes) were used. The stains were visualized by fluorescence microscopy. (3055)

Notes: A549 cells were grown to 80% confluence before being exposed to smoke or sham air for 4, 6 or 22 hours. Cells were harvested by trypsinization and viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3165)

Notes: Using the SV Total RNA Isolation System before RT-PCR, expression levels of AtGGT-IB were compared in flowers, leaves, stems, and root tissues of wildtype and era1-2 Arabidopsis plants. First-strand synthesis was performed with M-MLV Reverse Transcriptase using 500ng of RNA and an equal amount of oligo(dT). A 1:10 dilution of this product was used for amplification. (2855)

Notes: Cells were seeded in 96-well plates at 1 x 10⁴ cells/ml and exposed to arsenic trioxide, emodin, or the two-drug combination for 2–3 days with daily change of drug-containing medium. After treatment, cell viability was assayed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3151)

Notes: This paper describes the use of RQ1 RNase-Free DNase to remove DNA from TRIZOL- purified total RNA. The researchers describe stopping RQ1 RNase-Free DNase reactions with RQ1 RNase-Free DNase Stop Solution and heating the reactions at 64°C before using the samples in RT-PCR.  (3187)

Notes: H69AR cells were seeded 10⁴ into wells of a 96-well microtiter plate. After a 24-hour incubation, the medium was replaced with new media with doxorubicin (DOX), liposomes containing DOX, and/or sense or antisense oligonucleotides. After a 48 hour incubation, the plates were assayed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3161)

Notes: The CellTiter-Glo^® Luminescent Cell Viability Assay was used to examine the proliferation of BEAS-2B (immortalized human bronchial epithelial (HBE)) cells after treatment with various concentrations of erlotinib, an inhibitor of the mitogenic effects of EGF. For these experiments, 3,000/well were seeded in 96-well plates and incubated for 72 hours with various concentrations of erlotinib. Other lung cell carcinoma cell lines (A549, H226, H358, and H441) were also tested for proliferation in the presence of erlotinib using the CellTiter-Glo^® Assay system. The researchers used a luciferase construct that contained the cyclin D1 promoter to test the effects of erlotinib on cyclin d1 regulation. This construct, along with the pRL-TK vector, were co-transfected into BEAS-2B cells. Groups of cells were then treated with or without EGF and erlotinib and assayed for luciferase activity using the Dual-Luciferase^® Reporter Assay System.  (3258)

Notes: DNA was purified from blood stains and buccal swabs with DNA IQ™ System and two other comparative methods. A high throughput AluQuant^® assay on the BioMek^® 2000 was compared to a quantiblot method for quantifying human genomic DNA. DNA samples extracted with the DNA IQ™ System had less variability than the quantiblot method. The AluQuant^® System showed a similar level of sensitivity, reproducibility and precision compared to the quantiblot method.  (3007)

Notes: Cells were exposed to medium of 300, 540, and 620 mosmol/kg of H₂O for 0.5, 2, 6, 12 and 24 hours. Cell integrity was assayed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3160)

Notes: In this study, the CellTiter-Glo^® Luminescent Cell Viability Assay was used to analyze Jurkat cell viability after exposure to recombinant mouse galectin-1β (Gal-1β) or galectin-1α (Gal-1α). Data is presented as percent viable cells. Gal-1α was shown to have a more detrimental effect on cell viability.  (3172)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify genomic DNA from Yersinia pestis Kimberley53 strain.  The isolated genomic DNA was digested with restriction endonucleases and then used in PCR amplifications. The amplified products were sequenced to verify the presence of a transposon insertion site.  (3103)

Notes: This paper describes use of RNA interference (RNAi) to screen the genome of Drosophila melanogaster for genes affecting cell growth and viability. 19,700 double stranded RNAs were used to screen approximately 91% of the genome. The screen consisted of transfection of approximately 12,000 Kc₁₆₇ or S2R+ cells per well of a white 384-well plate with dsRNA. After a 5-day incubation, cell viability was assayed using the CellTiter-Glo® Luminescent Cell Viability Assay and a Molecular Dynamics Analyst HT. The authors report finding 438 target genes that affected cell growth or viability. (2843)