Introduction to Optimization of Cellular Transfection and Transduction
Optimization of cellular transfection and transduction includes choosing a protocol, determining the appropriate mass of plasmid/virus, and evaluating the optimum time after transfection/transduction for the best expression of the construct of interest. Using fluorescent reporters and non-destructive in situ imaging on Celigo cell imaging cytometer, these parameters can be rapidly optimized by repeated imaging of one set of wells or flasks over a period of time. Accurate whole-well bright field cell counting generates cell-based data normalized to absolute cell numbers.
- Plate 4 x 105 cells in a 6 well plate.
- Thaw virus vector stock at room temperature and store stock on ice
- Prepare a serial dilution ranging from 10-1 to 10-4
- Add 1 ml of diluted virus to pre-plated cells and incubate at 37°C
- Examine cells for GFP expression after 2 days of incubation
- Viral titer is determined by the percent of eGFP positive cells
Fast and Easy In-plate Analysis of Lentiviral Transduction Efficiency
- HEK293T cells were seeded and grown to 80 percent confluence
- Lentivirus was added to the wells at a 10-fold serial dilution
- After incubation, the Celigo was used to acquire bright field images and GFP images for each well and analyze the data
Bright field, fluorescent, and counted images of lentiviral titration for transduction
Images show captured bright field images for each dilution (bottom row), GFP positive cells for each dilution (top row) and Celigo software identified and counted (cells outlined in red) GFP positive cells (middle row).