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Plate Reader (plate + reader)
Kinds of Plate Reader Selected AbstractsA Mass Spectrometry Plate Reader: Monitoring Enzyme Activity and Inhibition with a Desorption/Ionization on Silicon (DIOS) PlatformCHEMBIOCHEM, Issue 7 2004Zhouxin Shen Dr. Abstract A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications. [source] Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresisELECTROPHORESIS, Issue 5-6 2006Shaobo Zhou Abstract We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24,h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54,protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10,dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in,vivo, so long as sufficient quantities of radioactive tracer are used. [source] Inkjet Printing of Luminescent CdTe Nanocrystal,Polymer Composites,ADVANCED FUNCTIONAL MATERIALS, Issue 1 2007E. Tekin Abstract Inkjet printing is used to produce well-defined patterns of dots (with diameters of ca.,120,,m) that are composed of luminescent CdTe nanocrystals (NCs) embedded within a poly(vinylalcohol) (PVA) matrix. Addition of ethylene glycol (1,2,vol,%) to the aqueous solution of CdTe NCs suppresses the well-known ring-formation effect in inkjet printing leading to exceptionally uniform dots. Atomic force microscopy characterization reveals that in the CdTe NC films the particle,particle interaction could be prevented using inert PVA as a matrix. Combinatorial libraries of CdTe NC,PVA composites with variable NC sizes and polymer/NC ratios are prepared using inkjet printing. These libraries are subsequently characterized using a UV/fluorescence plate reader to determine their luminescent properties. Energy transfer from green-light-emitting to red-light-emitting CdTe NCs in the composite containing green- (2.6,nm diameter) and red-emitting (3.5,nm diameter) NCs are demonstrated. [source] Immunoliposomes Sandwich Fluorometric Assay (ILSF) for Detection of Escherichia coli O157:H7JOURNAL OF FOOD SCIENCE, Issue 6 2004Sungsu Park ABSTRACT: We report the development of automated flourometric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating fluorophore as an analytical reagent. Thiolated antibodies (anti- E. coli O157:H7) were coupled to malemide-tagged liposomes encapsulating dye. To automate the assay, a fluorescence plate reader was included in the assay system to detect fluorophore released from lysed liposomes in a microplate. The detection limit of the current assay with pure cultures of the serotype was about 104 colony-forming units (CFU)/mL. The assay can detect E. coli O157 in ground beef samples inoculated with as few as 0.8 CFU/mL after a 12-h enrichment. These results demonstrate the feasibility of using fluorophore-encapsulated immunoliposomes in a microtiter plate for the rapid and automated detection of molecules with multivalent antigenic sites. [source] Ink-Jet Printing of Luminescent Ruthenium- and Iridium-Containing Polymers for Applications in Light-Emitting DevicesMACROMOLECULAR RAPID COMMUNICATIONS, Issue 4 2005Emine Tekin Abstract Summary: Defined films of luminescent ruthenium(II) polypyridyl-poly(methyl methacrylate) (PMMA) and iridium(III) polypyridyl-polystyrene (PS) copolymers could be prepared by ink-jet printing. The copolymers were deposited on photoresist-patterned glass substrates. Films as thin as 120 nm could be printed with a roughness of 1 to 2%. In addition, the film thickness could be varied in a controlled way through the number of droplets deposited per unit area. The topography of the ink-jet printed films was analyzed utilizing an optical profilometer. The absorbance and emission spectra were measured using fast parallel UV-vis and fluorescence plate reader. Photo of the solutions of luminescent ruthenium (left) and iridium (right) containing polymers in a glass microtiter plate (top). The subsequently prepared films using ink-jet dispensing techniques are shown below. [source] AlamarBlue bioassay for cellular investigation of UV-induced crystalline lens damageOPHTHALMIC AND PHYSIOLOGICAL OPTICS, Issue 4 2003Olanrewaju M. Oriowo Abstract Purpose: The use of the alamarBlue fluorescence dye for cellular study of UV-induced photodamage in cultured ocular lenses was examined by comparing the results from the fluorometric assay to lens optical quality using a scanning laser system to measure the focal lengths of the lenses following UVB treatment. Methods: Excised porcine lenses were cultured in M199 supplemented with 1% antibiotics and 4% porcine serum. After 1 week of pre-incubation at 37°C, baseline measurements were taken. Treated lenses were irradiated with a range of UVB radiant exposures from 0.019 to 0.076 J cm,2. The lenses were maintained for a further 4 weeks, with measurements carried out every 48 h in the first 9 days post-UVB treatment and then once each week. At each measurement session, treated and control lenses were transferred into a 24-well plate, one lens per well containing the assay. The lenses were incubated for 50 min, after which fluorescence readings were taken with a plate reader. Results: Analyses showed significant (p < 0.05) inhibition of lens metabolic activity and optical function in the 0.038 and 0.076 J cm,2 UVB treated lenses. Lenses treated with 0.019 J cm,2 UVB did not exhibit any photodamage. Conclusions: These results suggest that the alamarBlue assay is useful for the in vitro study of UV-induced lens damage. The decrease in the capacity of treated lenses to reduce alamarBlue over time confirms that UVB photo-oxidation can cause diminution of viable lens epithelial and fibre cells. The results also suggest that the energy threshold for broadband UVB induced cataractogenesis in vitro ranges between 0.019 and 0.038 J cm,2. [source] Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2003Anima Ghosal Abstract Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15,35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N -diethyl- N -methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions. Copyright © 2003 John Wiley & Sons, Ltd. [source] A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicineBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2007H Tian Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called ,caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery. British Journal of Pharmacology (2007) 150, 321,334. doi:10.1038/sj.bjp.0706988 [source] Tumor,stromal interactions with direct cell contacts enhance proliferation of human pancreatic carcinoma cellsCANCER SCIENCE, Issue 12 2009Hayato Fujita Pancreatic ductal adenocarcinoma is often characterized by an abundant desmoplastic stroma that is partially induced by activated pancreatic stellate cells (PSCs). Indirect co-culture has often been used to investigate the effects of cancer,stromal interactions on the proliferation of cancer cells, but the effects of cell,cell adhesion and juxtacrine signaling between cancer and stromal cells cannot be evaluated using this method. This study aimed to establish a simplified direct co-culture system that could be used to quantify populations of cancer cells in co-culture with PSCs, and to evaluate the effects of direct cell contact on the proliferation of cancer cells. We established three green fluorescent protein (GFP)-expressing pancreatic cancer cell lines and were able to quantify them with high reliability and reproducibility, even when co-cultured directly with PSCs, using a color plate reader. We assessed the differential effects of direct and indirect co-culture with PSCs on the proliferation of cancer cells, and found that the proliferation of GFP-expressing pancreatic cancer cell lines was dramatically enhanced by direct co-culture with PSCs, compared with the indirect co-culture system. We also found that direct co-culture of cancer cells and PSCs activated the Notch signaling pathway in both cell types. Direct cell contact between cancer cells and PSCs plays an important role in the control of cancer cell proliferation, and is essential to the understanding of tumor,stromal interactions. (Cancer Sci 2009; 100: 2309,2317) [source] The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interactionCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2010M. Choi Summary Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 ± 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0·1 µg/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6·25 to 100 µM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 µM (inhibition to 42 ± 4% with compound 27519 and to 47 ± 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-,-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1. [source] | ||||||||||||||||