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Seminar : Utilizing a Novel Engineered Shrimp-Derived Luciferase to Enable Real-Time In Vitro Measurement of Cell Viability and Apoptosis using a Plate-Reading Luminometer

Join the free seminar conducted by Dr. Terry Riss of Promega Corporation. Terry will speak about Promega's novel engineerd shrimp-derived luciferase and how research can hugely benefit from it.

  • Location: Pivot Park, Panoramaroom at Henry Chesbrough building Pivot Park - Oss.
  • Date: Monday, January 16, 2017
  • Time:  4:00 PM–5:00 PM (CET: Amsterdam, Berlin, Stockholm)
Free registration »

Contact: nikki.minnebo@promega.com

Join the complimentary onsite seminar:

Utilizing a Novel Engineered Shrimp-Derived Luciferase to Enable Real-Time In Vitro Measurement of Cell Viability and Apoptosis using a Plate-Reading Luminometer

The program will be presented by Terry Riss, PhD, Global Strategic Manager Cell Health at Promega Corporation

Abstract:

We have engineered two versions of a shrimp-derived luciferase to develop new in vitro assay technologies for toxicological research. The first version of this new technology uses an engineered luciferase and a pro-substrate as a reagent to monitor cell viability in real-time by measuring the conversion of the pro-substrate into a luciferase substrate which is limited to live cells. Viable cell number is monitored by recording luminescence repeatedly from the same sample of cells for days, providing kinetic information on the status of the culture. The real-time viability assay also provides a new tool to enable repeated dose toxicity experiments extending for weeks. Assay reagent can be delivered to cultures and viable cell number recorded just prior to the normal schedule of changing culture medium and replenishing the toxin of interest. The reagents are well tolerated by live cells which enables subsequent multiplexing of other assays such as extraction of RNA to analyze gene expression to identify specific stress response pathways. The second version uses small and large fragments of luciferase that have been engineered as fusion proteins linked to annexin V for detecting of apoptosis in real-time using a plate-reading luminometer. The individual annexin V-luciferase fragment fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence in culture medium or in the presence of non-apoptotic cells; but, when the annexin V-luciferase fragment fusion proteins bind in close proximity to phosphatidylserine exposed on the surface of apoptotic cells, the luciferase fragments can reconstitute an active enzyme and generate a luminescent signal. The annexin v-luciferase fragment fusion proteins and a luciferase substrate are combined to form a reagent that is added directly to cells in culture to create a homogeneous protocol that does not require cell washing steps typically used with fluorescent annexin V binding assays. Monitoring luminescence over time shows the onset of apoptosis precedes secondary necrosis measured from the same sample by multiplexing with a non-permeable fluorogenic DNA binding dye to indicate membrane integrity. These new luciferase assay technologies to monitor cell viability and apoptosis in real-time from the same samples provide kinetic information not available from endpoint assays and greatly simplify assay protocols compared to using flow cytometry or high content imaging.

About the Presenter

Dr. Riss received a PhD in Cell Biology from the University of Illinois in 1978 in the field of mammary gland biology. He did postdoctoral work at the University of Texas Medical School in Houston purifying growth factors involved with breast cancer and developing serum-free media formulations to implement bioassays. Dr. Riss moved to the Biotechnology division of Schering Plough in 1988 to develop interleukin/cytokine bioassays to support investigational new drug filings. Dr. Terry Riss started the Cell Biology program at Promega Corporation in 1990 and has held several R&D and Project Management positions. Dr. Riss managed development of cell viability, cytotoxicity, apoptosis, and protease assay systems and also lead efforts to identify and promote multiplexing of cell-based assays to determine the mechanism of cell death. Dr. Riss now serves as Global Strategic Marketing Manager, Cell Health involved in outreach educational training activities. Dr. Riss participates in NIH study sections reviewing HTS grants and is co-editor of the cell culture assays section of the Assay Guidance Manual hosted by NIH.

 

 

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