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 handling of the product.
* An MOI of 50 to 60 ifu/cell in HeLa cells has been calculated as optimum for the EGFP-GCCR assay. Other assay conditions will vary, and these can be optimized by the end-user.
Assay protocol (fixed cell)
On the day of the assay, test compounds were prepared in assay buffer. Medium was removed from the cells and replaced with medium containing CS-FCS (100-μl/well). Test compounds (e.g., 200-nM dexamethasone) were added* (100-μl/well) and incubated for 30 to 60 min at 37 ºC, 5% CO2.
The solutions were decanted from the wells and fixing solution added (100-μl/well) followed by incubation for 15-min at room temperature. The fixing solution was decanted from all wells, followed by washing with PBS (200-μl/well). Finally, Hoechst nuclear dye (2.5 μM) in PBS was added (100-μl/well), followed by incubation for 20 min at room temperature. The plates were stored at 2–8 °C if imaging was not performed immediately.
Image acquisitions were performed on the IN Cell Analyzer 1000 (using a 10× objective and 360/40-nm (Hoechst) and 475/20-nm (EGFP) excitation filters and 535/20-nm emission filter), or an IN Cell Analyzer 3000 (excitation provided by 488-nm [EGFP] and 363-nm [Hoechst] laser lines and monitored through 535/45-nm and 450/65-nm emission filters).
Images from IN Cell Analyzer 1000 were analyzed using the Nuclear Trafficking Analysis Module or the Object Intensity Analysis Module to quantitatively measure the fluorescence intensity in the cytoplasm and the nucleus of each cell. The nuclear/cytoplasmic (Nuc/Cyt