Marker Gene Monthly Newsletter   

December, 2002

Volume 2, Number 12

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Cell Viability/Cytotoxicity Assay

Analysis of cell viability in cell culture is an important means of evaluating in vitro drug or environmental effects in cell-mediated cytotoxicity assays or for monitoring cell proliferation and health in cell culture.  Our new, sensitive Live:Dead Assay Kit (M0795) can be used with mammalian cells, bacteria and yeast cells in culture, and provides quick, accurate and quantitative analysis of cell viability using the two compounds, carboxyfluorescein di-acetate (M0011) and propidium iodide (M0793) as in a protocol that allows staining of up to 1000 microplates of cells.   Analyses by fluorescence microscopy, flow cytometry or by using standard microtiterplate formats are easily performed. The red and green fluorescence emissions are easily resolved.  For more information about this assay, see the references below:

• "Nucleic acid detection using non-radioactive labelling methods." Mansfield ES, Worley JM, McKenzie S.,E., Surrey, S., Rappaport, E., Fortina, P., Mol. Cell Probes 9:145-156 (1995).
• "Flow cytometric assessment of viability of lactic acid bacteria." Bunthof CJ, Bloemen K, Breeuwer P, Rombouts, F.M., Abee, T., Appl. Environ. Microbiol. 67:2326-2335 (2001).
• "Fluorescence staining and flow cytometry for monitoring microbial cells." Veal DA, Deere D, Ferrari B, Piper J, Attfield PV. J. Immunol. Methods 243:191-210 (2000).
• "Flow cytometry and cell sorting for yeast viability assessment and cell selection." Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D. Yeast 14:147-160 (1998).

Ecdysone-Inducible Promoter Systems for lacZ ß-Galactosidase and GFP.

The new Ecdysone-Inducible mammalian expression systems offer a new way of controlled expression in mammalian cell lines.  These systems are somewhat complicated, comprising a vector with an ecdysone-glucocorticoid responsive element (E/GRE) directly upstream from its promoter (located in front of the target gene insertion site), and a helper plasmid (pVgRXR) that encodes modified ecdysone (VgEcR) receptor and retinoid X-receptor (RXR) proteins. Host cells cotransfected with these two plasmids and expressing housekeeping levels of the VgEcR and RXR receptor proteins can be induced to overexpress target gene protein with addition of ecdysone, ponasterone A or muristerone A. Induction is mediated by the binding of these phytosteroids to VgEcR and subsequent association with RXR. This protein complex then recognizes the E/GRE enhancer and binds, and the VP16 transactivation domain then interacts with the vector promoter initiating target gene expression.  Some side-effects of the ecdysteroids in mammalian systems have been noted.  For more information about the ecdysone inducible systems see the following references:

• Luers, G.H., Jess, N., Franz, T., “Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system.” (2000) Eur. J. Cell Biol. 79:653-657.
•   No, D., Yao, T.P., Evans, R.M., “Ecdysone-inducible gene expression in mammalian cells and transgenic mice.” (1996) Proc. Natl. Acad. Sci. US 93: 3346-3351.

Caspase and Serine Protease (Serpase) Activation During Apoptosis

Many proteases are important in the apoptosis pathway that leads to programmed cell death.  New fluorescent reagents capable of measuring both common caspases (Caspase 3-9) as well as several serine proteases have been developed in the laboratory of Zbigniew Darzynkiewicz at the Brander Cancer Research Institute, New York Medical College, Valhalla, New York.  These fluorescent aspartate and serine protease probes act as inhibitors of these important enzymes, and are capable of labeling cells at various stages of apoptosis.  For more information about these new probes see the references below:

• Smolewski, .P., Bedner, E., Du, L., Hsieh T.,C., Wu J.,M., Phelps D.,J., Darzynkiewicz Z., “Detection of caspases activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry”, Cytometry (2000) 44:73-82.
• Bedner, E., Smolewski, P., Amstad, P., Darzynkiewicz, Z., “Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation”, Exp. Cell Res. (2000) 259:308-313.
• Grabarek, J., Du, L., Johnson, G.L., Lee, B.W., Phelps, D.J., Darzynkiewicz, Z., “Sequential Activation of Caspases and Serine Proteases (Serpases) During Apoptosis”, Cell Cycle 1(2):124-131

ELISA Detection of ß-Gal Using FDG.

Ultra-sensitive detection of antigen expression on cell surfaces can be obtained using a combination of antibodies, that includes an “enzyme-linked immunosorbent assay” (ELISA) sandwich using the ß-galactosidase enzyme and the sensitive fluorogenic substrate fluorescein di-b-D-galactopyranoside (FDG) (M0250) for detection.  This assay provides a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes can also be reduced tenfold for use with Terasaki microtest plates and as little as 5 microliters of test supernatant can be screened.  For examples of these techniques see the references below. 

• D. Baruch, H. Glickstein, Z.I. Cabantchik "Plasmodium falciparum: Modulation of Surface Antigenic Expression of Infected Erythrocytes as Revealed by Cell Fluorescence ELISA.". Exp. Parasitol. 73: 440 (1991). Sadler, A.M.,
• Krausa P., Marsh S.G., Heyes J.M., Bodmer J.G., "A MicroElisa Assay for Detection of Anti-HLA Activity of Mouse Monoclonal Antibodies using an Astroscan 2100 Automated Plate Reader.",J. Immunol. Meth. 149:11 (1992).
  
  

Suppliers of Reporter Assay Screening Systems.

A recent issue of “The Scientist” surveyed the leading suppliers of Reporter Assay systems.   Our easy to use, ultra-sensitive kits for Luciferase (M0626, Live Cell Luciferase Assay Kit), in vivo lac Z ß-Galactosidase Intracellular Detection (M0259) and FACS analysis of lacZ ß-Galactosidase (M0255) scored well against the competition.  Try these new kits for your important live cell assays.  Each kit comes with the reagents, standards and a detailed protocol to run 250-1000 assays.  See the link below for more information.

• Sussman, H.E., “Choosing the Best Reporter Assay” (2001) The Scientist 15(15):25, http://www.the-scientist.com/yr2001/jul/profile2_010723.html)
• http://www.the-scientist.com/yr2001/jul/profile_010723.html
  
  

New Catalog Will Be Available Soon.

The 2003-2004 edition of the Marker Gene catalog will be mailed out in mid-January.  Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products.  Be sure to add your name to our mailing list.  Please visit our Web site and fill out our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you.
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Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

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 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.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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