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Performance of Ad-A-Gene EGFP-Glucocorticoid Receptor (EGFP-GCCR): an adenoviral vector gene delivery system

alysis Modules provided for your IN Cell Analyzer instrument. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.


Additional materials required
Instrumentation
FACSCalibur™ system (Becton Dickson)

Culture medium
DMEM (Sigma), with 10% fetal bovine serum (FBS) (Sigma), 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)

Culture medium plus charcoal-stripped FCS (CS-FCS): DMEM (Sigma) with 10% CS-FCS (Sigma)

Test compounds
Dexamethasone (Sigma). 50-mM stock in ethanol; β-estradiol, RU486, progesterone, and prednisolone (Sigma). Stocks in ethanol or DMSO at suitable concentrations.

PBS
Dulbecco’s (GIBCO BRL)

Fixative
Formalin solution (10%), neutral-buffered, 4% (w/v) formaldehyde, (Sigma). Dilute two-fold in PBS.

Dye
10-mM Hoechst™ 33342 nuclear dye (Molecular Probes).

Cell lines
HeLa, MCF7, A431, A549 (ATCC), and HEK 293 stable (GFP-GCCR) (GCCR gene obtained by permission from GlaxoSmithKline, and modified in-house into viral gene construct)


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. The adenoviral vector product 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%
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