Protocols and Applications Guide (US Letter Size) - Promega

|||||||| 8Bioluminescence Reporters
C. Live Cell Substrates
Researchers strive to monitor cellular activities with as little
impact on the cell as possible. The endpoint of an
experiment, however, sometimes requires complete
disruption of the cells so that the environment surrounding
the reporter enzyme can be carefully controlled. Recently,
nondestructive live cell substrates were developed, which
allow monitoring of Renilla luciferase without cell lysis.
Renilla luciferase requires only oxygen and coelenterazine
to generate luminescence, providing a simple luciferase
system with which to measure luminescence from living
cells. Unfortunately, coelenterazine is unstable in aqueous
solutions and so it has been difficult and inconvenient to
measure Renilla luciferase. EnduRen and ViviRen Live
Cell Substrates have been designed to overcome this
difficulty and to easily generate luminescence from live
cells expressing Renilla luciferase. Because the Renilla
luciferase luminescence is generated from living cells, these
substrates are also ideal for multiplexing with assays that
determine cell number.
Normalizing Interfering Phenomena
When making correlations between experimental conditions
and the expression of a reporter gene, other events
associated with cell physiology may affect reporter gene
expression. Of particular concern is the effect of cytotoxicity,
which can appear as genetic downregulation when using
a single-reporter assay. Live cell assays that can be
multiplexed with a cell viability assay allow independent
monitoring of both reporter expression and cell viability
to avoid misinterpretation of the data (Farfan et al. 2004).
The use of multiplexed assays allow correlation of events
within cells, such as the coupling of target suppression by
RNAi to its consequences on cellular physiology (Hirose
et al. 2002).
The CellTiter-Glo® Luminescent Cell Viability Assay
provides a rapid and sensitive assay of cell viability based
on luminescent detection of cellular ATP. Because the
CellTiter-Glo® Assay uses a stabilized firefly luciferase in
its formulation, it cannot be directly combined with a
reporter assay for expression of firefly luciferase. However,
it can be readily combined with nondestructive reporter
assays of Renilla luciferase.
Expression of Renilla luciferase may be quantitated, or
continuously monitored, by adding EnduRen Substrate
to the culture medium. When reporter measurements are
completed, CellTiter-Glo® Reagent is added to the sample
to inactivate the Renilla luminescence and initiate the
ATP-dependent luminescence. Because the CellTiter-Glo®
Assay is an endpoint assay, further sample monitoring after
measuring cell viability is not possible.
Protocols & Applications Guide
www.promega.com
rev. 3/09
Additional Resources for Live Cell Substrates
Technical Bulletins and Manuals
TM244 EnduRen Live Cell Substrate Technical
Manual
(www.promega.com
/tbs/tm244/tm244.html)
TM064 ViviRen Live Cell Substrate Technical
Manual
(www.promega.com
/tbs/tm064/tm064.html)
TM259 pGL4 Luciferase Reporter Vectors Technical
Manual
(www.promega.com
/tbs/tm259/tm259.html)
Citations
Dinh, D.T. et al. (2005) Helix I of Beta-arrestin is involved
in postendocytic trafficking but is not required for
membrane translocation, receptor binding and
internalization. Mol. Pharmacol. 67, 375–82.
Type 1 angiotensin II receptor Renilla luciferase
(AT1R-Rluc), and beta-arrestin1 and 2 GFP fusion constructs
were transfected into COS-7 cells. The COS-7 cell cultures
were then activated with 100μM angiotensin II in the
presence of 60μM EnduRen Live Cell Substrate, and
BRET fluorescence readings were taken at 475 and 515nm
over a 1-hour period. The authors also describe analysis of
helix I mutants of β-arrestin1 and β-arrestin2 in similar
β-arrestin-GFP construct BRET studies. Data are displayed
as a ratio of fluorescence readings with both constructs
compared to fluorescence from the AT1R-Rluc construct
alone.
PubMed Number: 15523053
Promega Publications
PN090 Measuring Renilla luminescence in living
cells.
(www.promega.com
/pnotes/90/12727_10/12727_10.html)
PN089 pGL4 Vectors: A new generation of
luciferase reporter vectors.
(www.promega.com
/pnotes/89/12416_07/12416_07.html)
D. Luciferase Reporter Cell Lines
Luciferase reporter assays have been widely used to
investigate cellular signaling pathways and as
high-throughput screening tools for drug discovery (Brasier
et al. 1992, Zhuang et al. 2006) The GloResponse Cell Lines
contain optimized, state-of-the-art luciferase reporter
technology integrated into a cell line. The GloResponse
NFAT-RE-luc2P HEK293 Cell Line and CRE-luc2P HEK293
Cell Line are designed for rapid and convenient analysis
of cell signaling through the nuclear factor of activated
T-cells (NFAT) pathway or cAMP response pathways via
activation of a luciferase reporter gene. Activity of
PROTOCOLS & APPLICATIONS GUIDE 8-11