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Appl. Environ. Microbiol. 73, 4234-4242. Rapid engineering of bacterial reporter gene fusions by using Red recombination. 2008

Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M.

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag® reporter using the HaloTag® coding region from the pHT2 promoter. (3924)

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Biochem. Biophys. Res. Commun. 373, 48–52. Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display. 2008

Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

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Clin. Vaccine Immunol. 15, 418–424. Sequential analysis of Anaplasma phagocytophilum msp2 transcription in murine and equine models of human. 2008

Scorpio, D.G., Leutenegger, C., Berger, J., Barat, N., Madigan, J.E. and Dumler, J.S.

Notes: The authors examined the pattern of Anaplasma phagocytophilum msp2 expression, a gene that modulates with little immune pressure and has decreased virulence with prolonged in vitro passage. C57BL/6J mice were inoculated with HL-60 cells infected with low-passage (passage 5) or high-passage (passage 26). Blood samples were taken 2–21 days post-inoculation, and total RNA was isolated. The purified RNA was subjected to RT-PCR, cloned into the pGEM®-T Easy Vector, transformed and plated. Plasmids were purified using the Wizard® SV 96 Plasmid DNA Purification System, and the insert size analyzed after EcoRI digestion. The inserts were sequenced, aligned with A. phagocytophilum Webster strain msp2 references using ClustalX and the diversity of msp2 transcripts divided into low- or high-passage bacteria. (3975)

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J. Biol. Chem. 283, 23514–23. Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins. 2008

Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.

Notes: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

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J. Bacteriol. 190, 1649–1657. Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus. 2008

Flahaut, S., Vinogradov, E., Kelley, K.A., Brennan, S., Hiramatsu, K. and Lee, J.C.

Notes: The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System. (3977)

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J. Virol. 12, 5940–50. Sulfatide is required for efficient replication of influenza A virus. 2008

Takahashi, T., Murakami, K., Nagakura, M., Kishita, H., Watanabe, S., Honke, K., Ogura, K., Tai, T., Kawasaki, K., Miyamoto, D., Hidari, K.I., Guo, C.T., Suzuki, Y. and Suzuki, T.

Notes: Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being subcloned into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418. (3990)

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BMC Genomics 9, 315. The complete mitochondrial genome of the Antarctic springtail Cryptopygus antarcticus (Hexapoda: Collembola). 2008

Carapelli, A., Comandi, S., Convey, P., Nardi, F. and Frati, F.

Notes: To sequence the mitochondrial genome one of the most widespread and common collembolan species of Antarctica, springtail Cryptopygus antarcticus. Specimens were collected from Killingbeck I during a 2002 polar expedition and frozen in liquid nitrogen. The Wizard® SV Genomic Purification System was used to extract total DNA from the samples and the complete mitochondrial genome was amplified twice, first with universal primers and sequenced, and then using long PCR with specific primers. The long PCR products were mechanically sheared, blunt end repaired and purified using the Wizard® SV Gel and PCR Clean-Up System. The fragments were then cloned, transformed and sequenced. (3976)

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Endocrinology 149, 2306–12. The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn. 2008

Vergnano, A.M., Ferrini, F., Salio, C., Lossi, L., Baratta, M. and Merighi, A.

Notes: The authors investigated the expression pattern of type 1a growth hormone secretagogue receptor (type 1a GHSR), a receptor for ghrelin. In situ RT-PCR was performed on paraformaldehyde-fixed, paraffin-embedded mouse spinal cord tissue. Prior to in situ RT-PCR, tissue sections were treated with proteinase K and triethanolamine, then dewaxed. Reverse transcription was performed using the Reverse Transcription System and oligo (dT)15 primers; followed by amplification using the PCR Master Mix in the presence of 11-digoxygenin-dUTP (1mM). Amplification products were detected using an anti-digoxygenin, alkaline phosphatase-conjugated goat antibody and nitro blue tetrazolium/5-bromo-3-indolylphosphate-p-toluidine salt (NBT/BCIP). (3968)

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Appl. Environ. Microbiol. 74, 2288–97. The genomes of the non-clearing-zone-forming and natural-rubber-degrading species Gordonia polyisoprenivorans and Gordonia westfalica harbor genes expressing Lcp activity in Streptomyces strains. 2008

Bröker, D., Dietz, D., Arenskötter, M. and Steinbüchel, A.

Notes: Natural rubber-degrading bacteria fall into two categories: those forming clearing zones on latex overlay plates and those that do not. To investigate this degradation process, the authors amplified latex-clearing protein (lcp) homologs from non-clearing-zone-forming bacteria using degenerate PCR primers based on lcp sequences from clearing-zone forming species. The 3´ region of the lcp gene in G. westfalica was amplified by nested PCR using biotinylated primers, and the amplified products were cloned in the pGEM®-T Easy Vector and sequenced using universal M13 forward and reverse primers. (3907)

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J. Biol. Chem. 283, 16868–75. The hairless phenotype of the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA containing five basic keratin genes. 2008

Nanashima, N., Akita, M., Yamada, T., Shimizu, T., Nakano, H., Fan, Y. and Tsuchida, S.

Notes: The mutation responsible for the hairless phenotype was linked to a 80kb deletion of chromosome 7q36. Because many basic keratin genes are located at 7q36, the authors examined keratin gene expression in the Hirosaki hairless rat using RT-PCR. Expression of kb21, kb23 and kb26 was not detected, whereas other basic keratin genes were expressed. RT-PCR was performed using the AccessQuick™ RT-PCR System and 0.5µg of total RNA isolated from rat skin for 21–30 cycles. (3887)

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Genetics 179, 177-192. The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii. 2008

Wang, Y., Ladunga, I., Miller, A.R., Horken, K.M., Plucinak, T., Weeks, D.P. and Bailey, C.P.

Notes: These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM®-T Easy Vector before transfer into an expression vector. (3875)

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Eukaryot. Cell 7, 1965–1979. Transcriptome for photobiological hydrogen production induced by sulfur deprivation in the green alga Chlamydomonas reinhardtii. 2008

Nguyen, A.V., Thomas-Hall, S.R., Malnoë, A., Timmins, M., Mussgnug, J.H., Rupprecht, J., Kruse, O., Hankamer, B. and Schenk, P.M.

Notes: The authors analyzed the transcriptional activity of wild-type Chlamydomonas reinhardtii cultures sampled at different time points during the aerobic and anaerobic phase of the photobiological hydrogen production process under sulfur-depleted conditions. C. reinhardtii were grown in photobioreactors, carefully extracted, centrifuged and flash-frozen in liquid nitrogen. RNA was purified using the SV Total RNA Isolation System following the plant centrifugation protocol without sample grinding. The eluted RNA was quantitated and integrity checked by gel electrophoresis and qRT-PCR. Total RNA was used to synthesize labeled cDNA using the ChipShot™ Indirect Labeling and Clean-Up System. The labeled cDNA was used for probing microarrays. (4022)

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Appl. Environ. Microbiol. 74, 1886–91. Use of Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections. 2008

Luce-Fedrow. A., Von Ohlen, T., Boyle, D., Ganta, R.R. and Chapes, S.K.

Notes: The authors infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples. (3888)

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Exp. Biol. Med. 232, 1195–1203. 13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro. 2007

O'Reilly, K.C., Trent, S., Bailey, S.J. and Lane, M.A.

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

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Assay Drug Dev. Technol. 5, 237–245. A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP 2007

Kumar, M., Hsiao, K., Vidugiriene, J. and Goueli, S.A.

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

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Mol. Biol. Cell 18, 2795–804. A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells. 2007

van Lith, M., Karala, A.R., Bown, D., Gatehouse, J.A., Ruddock, L.W., Saunders, P.T. and Benham, A.M.

Notes: PDILT is a protein disulfide isomerase (PDI) homolog under developmental control and is induced during puberty. To determine whether PDILT expression coincides with the first wave of spermatogenesis, the authors examined expression of PDILT in 2-, 15-, 30-, 45- and 58-day old rats using RT-PCR. Total RNA was isolated from rat testis, and 50ng was amplified using the AccessQuick™ RT-PCR System and primer pairs specific for PDILT, PDI and calmegin, a testis-specific chaperone that is expressed upon the appearance of spermatocytes. Primer pairs were designed to span introns to avoid amplification of genomic DNA. PCR products were analyzed on a 1% agarose gel. Results showed that PDILT mRNA was first detected during the onset of spermatogenesis. (3765)

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Development 134, 2889–2894. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. 2007

Stavridis, M.P., Lunn, J.S., Collins, B.J. and Storey, K.G.

Notes: The authors studied the role of the Erk1/2 signaling pathway during neural specification in mouse embryonic stem (ES) cells. Undifferentiated ES cells express high levels of the pluripotent marker Nanog but do not express fibroblast growth factor (Fgf5), an early marker of differentiation of ES cells, or Sox1, an early neural transcription factor gene. Using quantitative PCR, levels of Nanog, Fgf5 and Sox1 mRNA were quantitated during ES differentiation in the presence and absence of a MEK inhibitor. Prior to quantitative PCR, 1µg of total RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase. By measuring these mRNA levels, the authors determined that inhibition of the Erk1/2 pathway blocked ES differentiation. (3726)

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J. Biol. Chem. 282, 29211–21. A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits. 2007

Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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Am. J. Pathol. 171, 19–31. A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. 2007

Brehm, R., Zeiler, M., Rüttinger, C., Herde, K., Kibschull, M., Winterhager, E., Willecke, K., Guillou, F., Lécureuil, C., Steger, K., Konrad, L., Biermann, K., Failing, K. and Bergmann, M.

Notes: To study the role of connexin42 (cx43) in testis development, the authors generated a conditional cx43 knockout mouse, which lacked the cx43 gene in Sertoli cells. To confirm that the cx43 gene was deleted in these mice, PCR was performed using primers specific to cx43, 1X Colorless GoTaq® Flexi Reaction Buffer, 2mM MgCl2, dNTPs and 0.15µl of GoTaq® DNA Polymerase. Tissue-specific deletion of cx43 was confirmed by amplifying RNA isolated from mouse testis, heart and tail was also confirmed using the same PCR components. (3713)

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Cancer Res. 67, 1472-1486. Adaptation of energy metabolism in breast cancer brain metastases. 2007

Chen, E.I., Hewel, J., Kreuger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R., and Felding-Habermann, B.

Notes: This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis. (3613)

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J. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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Forensic Sci. Int. 170, 68–72. Allele frequency distribution for 21 autosomal STR loci in Bhutan. 2007

Kraaijenbrink, T., van Driem, G.L., Tshering of Gaselô, K. and de Knijff, P.

Notes: The authors determined the allele frequencies for 21 autosomal STR loci in 936 individuals from the Royal Kingdom of Bhutan using the AmpFlSTR® Identifiler® kit, PowerPlex® 16 System and the F13A01, FESFPS, F13B, LPL (FFFL) Multiplex. DNA was extracted from whole blood samples using the Autopure LS® kit and amplified as directed by the manufacturers. Amplified products were detected using an ABI PRISM® 3100 Genetic Analyzer. (3822)

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Forensic Sci. Int. 168, 227–231. Allele frequency distribution for 21 autosomal STR loci in Nepal. 2007

Kraaijenbrink, T., van Driem, G.L., Opgenort, J.R., Tuladhar, N.M. and de Knijff, P.

Notes: Allele frequency data for 21 autosomal loci was studied in 953 unrelated individuals belonging to 12 major Nepalese groups. DNA was extracted from whole blood using the Autopure LS® kit, then amplified using the PowerPlex® 16 System, AmpFlSTR® Identifiler® kit or F13A01, FESFPS, F13B, LPL Multiplex (FFFL) Multiplex. Amplification products were analyzed using the ABI PRISM® 3100 Genetic Analyzer. Several new alleles not reported in the NIST short tandem repeat database were detected in this population. (3811)

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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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