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General Considerations for Successful Transfection Experiments

Cells

General
Keep an eye on your cells; ensure they are in good condition and set a suitable plating protocol for optimal cell density from the beginning to the end of transfection.

Dividing versus Non-dividing Cells
Dividing cells tend to be more accessible for uptake and expression of foreign DNA compared to quiescent cells. Mitogenic stimuli (e.g., virus transformation, growth factors, conditioned media, and feeder cells) are often used to activate primary cells.

Adherent versus Suspension Cells
Transfection efficiencies differ significantly between adherent and suspension cells. It is speculated that the limiting step is the uptake by endocytosis; however, a plausible mechanistic explanation on a molecular level does not yet exist. Therefore, the search for more efficient transfection reagents is mainly empirical.

Splitting Protocol
Before splitting, adherent cells must be trypsinated in order to remove them from the substrate. This routine step means a severe obstruction of normal cellular functions. Differences in the splitting protocol (e.g., extension of trypsination, inactivation of trypsin, and time until transfection starts) could have an impact on transfection experiments.

Passage Number
Cell lines tend to be unstable. As a result, their features may change over time in culture. The passage number indicates how often a cell line has been split (normally within one lab). In most cases, the exact passage number existing prior to establishment of the line is unknown. Different culture conditions could lead to clonal selection. Cell lines with the same name could thus significantly differ with respect to physiology and morphology (and transfectability). Generally, cells are more difficult to transfect the first passage or two out of the freezer. Thereafter, with some cell lines the transfection efficacy remains relatively constant for over 100 passages. With other cell lines, transfection efficacy may either increase or decrease with passage.

Cell Number (Grade of Confluency)
Cell lines divide exponentially when there is space on the substrate (tissue culture dish). The growth rate is negatively affected by cell density (contact inhibition), depletion of nutrients, and metabolic end products (e.g., pH). The extent of reporter gene expression is directly correlated with the cell number at the beginning of transfection and the growth rate prior to cell lysis.


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