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

) ratio is the population-averaged ratio of sampled nuclear and cytoplasmic intensities measured in the EGFP signal channel.

* Time-controlled dispensing of compound solutions to the cells can be avoided by completing the assay using live cells and then fixing the cells prior to imaging. Cells should be fixed at the peak translocation time point. Fixing of cells reduces the handling requirements for post-assay material to BSL-1 categorization.


Assay protocol (kinetic live cell)
Test compounds (e.g., 200-nM dexamethasone containing 2.5-μM Hoechst nuclear dye) were added at 100-μl/well to cells in CS-FCS medium (100 μl) that had been transduced 24 h earlier as described previously. Plates were incubated for 30–60 min at 37 ºC, 5% CO2.

Cells were imaged on an appropriate detection instrument, using the image acquisition method as described above for the fixed-cell assay protocol. Analysis of images also followed the method outlined above for the fixed-cell assay protocol.

Note: at all stages of the live-cell assay, the assay plate must be contained within BSL-2 level facilities.


Results
Viral transduction efficiency measurement from flow cytometry data
The primary step to setting up a functional cellular assay involving adenoviral transduction is to determine the optimal MOI by titrating into target cells. We have made use of flow cytometry to quantitate the EGFP signal from cells transduced with the EGFP-GCCR adenoviral vector. Analysis of data allowed calculation of the transduction efficiency in HeLa cells at three MOI loadings (Table 1). HeLa cells were set up at 1 × 106 cells/well of a 6-well plate and virus was added at 0 (control), 25, 50, and 100 MOI. The plates were stored for 24 h and then the cells were trypsinized and resuspended in PBS followed by gated analysis on a FACSCalib
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