New Bacterial Quantitation Assay.

• Russo-Marie F, Roederer M, Sager B, Herzenberg L, (1993) " b-Galactosidase Activity in Single Differentiating Bacterial Cells." Proc. Natl. Acad. Sci. USA 90:8194.
• Nelis H, Van Poucke S, (2000) "Enzymatic detection of coliforms and Escherichia coli within 4 hours." Water, Air, and Soil Pollution 123(1-4): 43-52.
• Dreier J, Breitmaier EB, Gocke E, Apfel, CM, Page, MGP (2002) "Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay." Mutation Research 513(1-2): 169-182.
• Rowland B, Purkayastha A, Monserrat C, Casart Y, Takiff H, McDonough KA (1999) “Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species” FEMS Microbiol. Lett. 179(2): 317-25.
• Kevin L. Griffith and Richard E. Wolf, Jr. (2002) “Measuring beta-Galactosidase Activity in Bacteria: Cell Growth, Permeabilization, and Enzyme Assays in 96-Well Arrays”  Biochemical and Biophysical Research Communications 290: 397–402.
• Miller, J. H. (1972) "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory Press, Cold Spring Harbor
• Wutor VC, Togo CA, Pletschke BI, (2009) "Suitability of total coliform β-D-galactosidase activity and CFU counts in monitoring faecal contamination of environmental water samples"Water SA 35(1): 85-88.
  

Rhodamine 123 Multiple Drug Resistance Assay.

Resistance to chemotherapy was recognized early on as a serious impediment to cancer treatment. Tumor cells quickly gained resistance to specific compounds and also frequently demonstrated cross-resistance to a broad spectrum of structurally unrelated agents. This so-called multiple drug resistance (MDR) has been found to be due to the over expression of active transporters of certain classes of drugs. In addition, there is some evidence that MDR can be caused in part by reduced plasma membrane permeability to the cytotoxic compounds. Overcoming MDR has therefore become a major challenge for chemotherapy protocols. The fluorescent dyes Hoechst 33342 and rhodamine 123 (R123) are known to bind to the MDR transporters ABCG2 and ABCB1, respectively. A common test for MDR has been developed in which the cells are loaded with the fluorescent dye Rhodamine 123 (M0542) and the active transport of this dye out of the cells (efflux) is measured. R123 is a cell-membrane permeable fluorescent dye that has been found to bind to the active transporters in a manner similar to many chemotherapeutic drugs.

To stain cells, R123 (M0542)(50 to 200 ng/mL) is added to the extracellular environment (media) where it crosses into cells and accumulates in the cells' cytosol and mitochondria. Once cells have reached a steady state level of R123, the cells are then washed and prepared for measurement. If a cell is exhibiting multi-drug resistance, the R123 is rapidly pumped from the cell. The rate of loss from individual cells or a suspension of cells, loaded with R123 and washed, can then be examined using either traditional flow cytometer measurements or in a microtiterplate assay. With a traditional flow cytometer, the distribution of fluorescence at several time points is acquired. Studies with various tumor cell types have been measured and are available in the literature. For more information about these assays, please see the references below or visit our website.

For more information about these methods and protocols, please visit our website or see the references below.

• Vera S. Donnenberg and Albert D. Donnenberg (2005) "Multiple Drug Resistance in Cancer Revisited: The Cancer Stem Cell Hypothesis." J. Clin. Pharmacol. 45:872-877.
• Ludescher C, Gattringer J, Drach J, Hofmann J, Grunicke H, (1991) "Rapid functional assay for the detection of multidrug-resistant cells using the fluorescent dye rhodamine 123." Blood 78: 1385-1387.
• Tan B, Piwnica-Worms D, Ratner L. (2000) "Multidrug resistance transporters and modulation." Curr. Opin. Oncol. 12:450-458.
• Tucci M, Quatraro C, Dammacco F, Silvestris F (2009) "Role of active drug transporters in refractory multiple myeloma. Curr Top Med Chem 9:218-224.
• Altenberg GA, Vanoye CG, Horton JK, Reuss L (1994) "Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane." Proc. Natl. Acad. Sci. USA 91:4654-4657.
• Chen LB, (1988) "Mitochondrial membrane potential in living cells." Ann. Rev. Cell Biol. 4:155-181.

Yeast Live:Dead Assay

Yeast cells have become a popular cloning system for expression of eukaryotic proteins in high levels. Because many vectors, expression systems, marker genes and other reagents have been developed for yeast transformation, including those offering mammalian-type post-translational modifications of the expressed proteins, these new systems have gained acceptance in the molecular biology community. However, monitoring transfection efficiency and viability of transformed cells is an important consideration for these techniques.

In order to measure viability, the yeast can be stained using the MarkerGene^TM Live:Dead Assay Kit (M0795). This kit takes advantage of the presence of esterases in viable yeast, which are a family of catabolic enzymes that cleave esters in a wide range of naturally occurring substrates intracellularly. They are ubiquitous to almost all living organisms. The esterase activity of living cells can be monitored using the fluorescent substrate 5(6)-carboxyfluorescein diacetate (M0011) contained in the M0795 kit . Upon cleavage, the fluorescent product 5(6)-carboxyfluorescein (green fluorescence color) (M0122) is released and activity measurements are easily obtained either in vitro, in cell preparations, or in culture. The kit also contains the DNA stain propidium iodide (M0793) that is membrane impermeant to live yeast cells, but can enter the nucleus of dead cells and stain the chromatin (red fluorescence color). Measurement of red versus green fluorescence therefore gives an accurate measurement of dead versus live cell number (viability). Yeast with intact cell membranes stain fluorescent green, whereas yeast with damaged membranes (dead cells) stain fluorescent red (form propidium iodide binding the chromatin/DNA/RNA).  For more information about these assays, please see the references below or visit our website.

• Breeuwer P, Drocourt JL, Bunschoten, Zwietering MH, Rombouts FM, Abee T (1995) "Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product." Appl Environ Microbiol. 61(4): 1614-1619.
• Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D.  (1998) "Flow cytometry and cell sorting for yeast viability assessment and cell selection." Yeast 14: 147-160.
• Oh KB, Matsuoka H (2002) "Rapid viability assessment of yeast cells using vital staining with 2-NBDG, a fluorescent derivative of glucose." Intl. J. Food Microbiol. 76(1-2): 47-53.
  

H1N1 Influenza A Carbohydrate Binding Profile .

The H1N1 (hemagglutinin 1, neuraminidase 1) subtype, or 2009 H1N1 strain influenza virus has emerged as an important human infection with increasing transmissability in the global population. The method by which several strains of the virus initiate infection has been predicted to be through the hemagluttinin type sialyl-oligosaccharides terminating in neuraminic acids. In particular HA-glycan structures terminating with N-acetylneuraminic acid linked to galactose (Neu5Aca2-6Gal and Neu5Aca2-3Gal) would be likely targets for the virus as these structures appear on human epithelial cells particularly in the upper and lower respiratory tract. Recent work from the laboratories of Ten Feizi and co-workers at the Glycosciences Laboratory at the Imperial College of London along with co-workers in Germany, Japan, Portugal and Spain have used new nitrocellulose-based carbohydrate microarrays to define the binding characteristics for six strains of the H1N1 virus, and found them to indeed locate predominantly to these carbohydrate structures on the cell surface of human cells. This data indicates that the H1N1 virus did not have to make any major changes in HA specificity in order to acquire human binding characteristics and may contribute to its sustained human to human transmission. The implications of this work for the development of targeting mechanisms for the virus and potential new treatment regimes are significant. For more information about these assays, please see the references below or visit our website.

• Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W, Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A, Matrosovich M, Feizi T (2009) Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray." Nature Biotechnol. 29(9): 797-799.
• Fukui S, Feizi T, Galustian C, Lawson AM, Chai W, (2002) "Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions." Nature Biotechnol. 20: 1011-1017.
• Palma AS, (2006) "Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides." J. Biol. Chem. 281: 5771-5779.
• Soundararajan V, Tharakaraman K, Raman R, Raguram S, Shriver Z, Sasisekharan V , Sasisekharan R (2009) "Extrapolating from sequence—the 2009 H1N1 'swine' influenza virus." Nature Biotechnol. 27: 510-513.

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Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects. 

Contract Research (CRO) and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays - High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
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