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Performance of Ad-A-Gene Cyclic AMP Response Element-Nitroreductase (CRE-NTR): an adenoviral vector gene delivery system

BS) (Sigma), 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)

Assay medium: DMEM (Sigma) without phenol red or FBS, containing 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)

Forskolin (Sigma)

Dulbecco’s (GIBCO BRL)

Formalin solution (10%), neutral-buffered, 4% (w/v) formaldehyde, (Sigma)

Hoechst™ 33342 nuclear dye (Molecular Probes)

96-well microplates (Greiner Bio One)

U2OS cell lines (ATCC)


Assay method in 96-well microplate format
On the day before the assay, suitable target cells in log-phase growth were detached by treatment with trypsin. Cell concentrations were adjusted to suitable levels with culture medium. Ad-A-Gene CRE-NTR vector was thawed by placing the tube on ice, added to the cell suspension, and mixed to provide the appropriate multiplicity of infection (MOI) for the assay*. The cell and virus suspension was dispensed at 200 μl/well into microplates and incubated for 24 h at 37 °C, 5% CO2.

Note: All adenoviral vector preparations are handled as BSL-2 category reagents. Local safety assessments are made before using the reagent. The product pack literature contains detailed instructions for product handling.

*An MOI of 25 ifu/cell in U2OS cells has been calculated as optimum for the CRE-NTR assay. Other assay conditions will vary and can be optimized by users.


Assay protocol (fixed-cell format)
On the day of the assay, test compound (10-μM forskolin) was prepared from stocks in assay buffer. Medium was removed from the cells, and the cells were washed with PBS. Test wells were treated with 90 μl of forskolin in assay medium; 90 μl of medium alone was added to control wells. Pla
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