( BS) (Sigma) 2-mM L-glutamine (Sigma) ...)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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