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#SLLU-200 SuperLight™ Luciferase Reporter Gene Assay Kit
Product name : SuperLight™ Luciferase Reporter Gene Assay Kit
Catalog number : SLLU-200
Supplier name : Bioassays
Data sheet: Ask more or other datasheet now !
More Details about
Bright bioluminescent reagent system for rapid quantitation of luciferase reporter gene expression in transfected cells and high-throughput drug screens.
Samples: cells etc.
Procedure: 2 min.
Size: 200 tests.
Detection limit: 2 fg luciferase.
The SuperLightTM Luciferase Reporter Gene Assay is based on the
quantitation of luciferase expression in mammalian, yeast or E. coil cells,
using luciferin and ATP as substrates. The reaction results in light
production which can be conveniently measured on a luminometer.
ATP + D-luciferin + O2 oxyluciferin + AMP + PPi + CO2 + light
This bioluminescent reporter gene assay is extremely sensitive and is
especially suitable for quantifying luciferase expression in recombinant
cells. This ultra-sensitive, homogeneous cell-based assay only requires
adding a single reagent to the cells and measuring the light intensity after a
short incubation step (2 minutes). Assays can be performed in tubes,
cuvettes or multi-well plates. All kit components are compatible with culture
media and with all liquid handling systems. With an extended luminescence
emission kinetics (half-life 40 min), the SuperLightTM luciferase assays are
especially suitable for high-throughput screening in 96-well, 384-well and
1536-well plates. In addition, the reagent provided in the kits has been
formulated for maximum sensitivity, reproducibility and long shelf-life.
Applications for this kit include gene regulation studies and high-throughput
screening of gene modulators.
High sensitivity and wide detection range: detection of as little of 2 fg
luciferase and as few as 4 cells. Plus, the emitted light is linear over seven
orders of magnitude.
Compatible with routine laboratory and HTS formats: assays can be
performed in tubes or microplates, on LJL Analyst, Berthold Luminometer,
Top-Count, MicroBeta counters, chemiluminescent image plate readers
(CLIPR/LeadSeeker). Assay reagents compatible with all liquid handling
Fast and convenient: homogeneous “mix-and-measure” assay allows
detection of luciferase levels within 10 minutes. The optimally combined
reagent system allows a single addition step, and simultaneous cell lysis
Robust and amenable to HTS: Z’ factors of 0.6 to 0.8 are observed in 96-
well and 384-well plates. Can be readily automated on HTS liquid handling
Gene Regulation: gene expression level, characterization of promoter
activity, modulation of gene expression by receptors, transcription factors
and small molecules.
Drug Discovery: high-throughput screen for gene modulators.
Storage conditions. The kit is shipped at ambient temperature. Store the
Reagent in the provided amber tube at -20°C and the Assay Buffer at 2-8°C.
Shelf life: 12 months after receipt.
This protocol can be downloaded online at www.bioassaysys.com.
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents.
Sample Type: cell line
References: McGannon CM et al (2010). Different classes of antibiotics differentially influence shiga toxin production. Antimicrob Agents Chemother. 54(9):3790-8.
Pubmed ID: 20585113
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20585113
Abstract: Shiga toxin (Stx) in Escherichia coli O157:H7 is encoded as a late gene product by temperate bacteriophage integrated into the chromosome. Phage late genes, including stx, are silent in the lysogenic state. However, stress signals, including some induced by antibiotics, trigger the phage to enter the lytic cycle, and phage replication and Stx production occur concurrently. In addition to the Stx produced by O157:H7, phage produced by O157:H7 can infect harmless intestinal E. coli and recruit them to produce Shiga toxin. To understand how antibiotics influence Stx production, Stx lysogens were treated with different classes of antibiotics in the presence or absence of phage-sensitive E. coli, and Stx-mediated inhibition of protein synthesis was monitored using luciferase-expressing Vero cells. Growth-inhibitory levels of antibiotics suppressed Stx production. Subinhibitory levels of antibiotics that target DNA synthesis, including ciprofloxacin (CIP) and trimethoprim-sulfamethoxazole, increased Stx production, while antibiotics that target the cell wall, transcription, or translation did not. More Stx was produced when E. coli O157:H7 was incubated in the presence of phage-sensitive E. coli than when grown as a pure culture. Remarkably, very high levels of Stx were detected even when growth of O157:H7 was completely suppressed by CIP. In contrast, azithromycin significantly reduced Stx levels even when O157:H7 viability remained high.
PMID: 20585113 [PubMed - indexed for MEDLINE] PMCID: PMC2935008
References: Zhao L, Haslam DB (2005). A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis. J Med Microbiol 54:1023–1030.
Pubmed ID: 16192432
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=16192432
Abstract: Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml(-1). The assay was adapted for use in 24-, 96- and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.
PMID:16192432 [PubMed - indexed for MEDLINE]
References: Gentry M et al (2007). Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection. Infection and Immunity 75(8): 3969-3978.
Pubmed ID: 17502386
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=17502386
Abstract: Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis.
PMID: 17502386 [PubMed - indexed for MEDLINE] PMCID: PMC1951971
References: Roupelieva M et al (2010). Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex. J Gen Virol. 91(5):1138-49.
Pubmed ID: 20089804
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20089804
Abstract: In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.
PMID: 20089804 [PubMed - indexed for MEDLINE]
Sample Type: vero cells
References: Saenz JB et al (2007). Identification and Characterization of Small Molecules That Inhibit Intracellular Toxin Transport. Infection and Immunity 75(9): 4552–4561.
Pubmed ID: 17576758
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=17576758
Abstract: Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.
PMID: 17576758 [PubMed - indexed for MEDLINE] PMCID: PMC1951202
Sample Type: cell line
References: Kulkarni AA et al (2010). Glycan encapsulated gold nanoparticles selectively inhibit shiga toxins 1 and 2. Bioconjug Chem. 21(8):1486-93.
Pubmed ID: 20669970
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20669970
Abstract: Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae cause life-threatening conditions that include hemolytic uremic syndrome (HUS), kidney failure, and neurological complications. Cellular entry is mediated by the B-subunit of the AB(5) toxin, which recognizes cell surface glycolipids present in lipid raft-like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These highly soluble glyconanoparticles were nontoxic to the Vero monkey kidney cell line and protected Vero cells from Stx-mediated toxicity in a dose-dependent manner. The inhibition is highly dependent on the structure and density of the glycans; selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved. Interestingly, natural variants of Stx2, Stx2c, and Stx2d possessing minimal amino acid variation in the receptor binding site of the B-subunit or changes in the A-subunit were not neutralized by either the Stx1- or Stx2-specific gold glyconanoparticles. Our results suggest that tailored glyconanoparticles that mimic the natural display of glycans in lipid rafts could serve as potential therapeutics for Stx1 and Stx2. However, a few amino acid changes in emerging Stx2 variants can change receptor specificity, and further research is needed to develop receptor mimics for the emerging variants of Stx2.
PMID: 20669970 [PubMed - indexed for MEDLINE] PMCID: PMC3024884
References: Fuller CA et al (2011). Shiga toxin subtypes display dramatic differences in potency. Infect Immun. 79(3):1329-37.
Pubmed ID: 21199911
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=21199911
Abstract: Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.
PMID: 21199911 [PubMed - indexed for MEDLINE] PMCID: PMC3067513
References: Cohen ME et al (2011). Antibody against extracellular vaccinia virus (EV) protects mice through complement and Fc receptors. PLoS One. 6(6):e20597
Pubmed ID: 21687676
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=21687676
Abstract: Protein-based subunit smallpox vaccines have shown their potential as effective alternatives to live virus vaccines in animal model challenge studies. We vaccinated mice with combinations of three different vaccinia virus (VACV) proteins (A33, B5, L1) and examined how the combined antibody responses to these proteins cooperate to effectively neutralize the extracellular virus (EV) infectious form of VACV. Antibodies against these targets were generated in the presence or absence of CpG adjuvant so that Th1-biased antibody responses could be compared to Th2-biased responses to the proteins with aluminum hydroxide alone, specifically with interest in looking at the ability of anti-B5 and anti-A33 polyclonal antibodies (pAb) to utilize complement-mediated neutralization in vitro. We found that neutralization of EV by anti-A33 or anti-B5 pAb can be enhanced in the presence of complement if Th1-biased antibody (IgG2a) is generated. Mechanistic differences found for complement-mediated neutralization showed that anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice.
PMID: 21687676 [PubMed - in process] PMCID: PMC3110783
Sample Type: cells
References: Zaliauskiene L et al (2010). Efficient gene transfection using novel cationic polymers poly(hydroxyalkylene imines). Bioconjug Chem. 21(9):1602-11
Pubmed ID: 20695432
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20695432
Abstract: A series of novel cationic polymers poly(hydroxyalkylene imines) were synthesized and tested for their ability to transfect cells in vitro and in vivo. Poly(hydroxyalkylene imines), in particular, poly(2-hydroxypropylene imine) (pHP), poly(2-hydroxypropylene imine ethylene imine) (pHPE), and poly(hydroxypropylene imine propylene imine) (pHPP) were synthesized by polycondensation reaction from 1,3-diamino-2-propanol and the appropriate dibromide. Electron microscopic examination demonstrated that the resulting polymers condensed DNA into toroid shape complexes of 100-150 nm in size. Transfection studies showed that all three polymers were able to deliver genetic material into the cell, with pHP being superior to pHPP and pHPE. pHP acted as an efficient gene delivery agent in a variety of different cell lines and outcompeted most of the widely used polymer or lipid based transfection reagents. Intravenous administration of pHP-DNA polyplexes in mice followed by the reporter gene analysis showed that the reagent was suitable for in vivo applications. In summary, the results indicate that pHP is a new efficient reagent for gene delivery in vitro and in vivo.
PMID: 20695432 [PubMed - indexed for MEDLINE]
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