Dear Prof. Khadijeh Jamialahmadi

You will have to determine the selection efficiency by evaluating the effect of the selection antibiotic on both un-transfected and transfected cells of a given cell line.

Before starting a transfection experiment, it is advisable to determine the optimal initial cell density for transfection and the selection antibiotic concentration for selection by performing a kill curve assessment. If both conditions are not well balanced, untransformed parental cells can survive, or transfected cells may be killed. However, this kill curve simply defines the ideal concentration of selection antibiotic needed to kill the un-transfected cells of a particular cell line in 7–10 days of selection. A kill curve provides no information on the efficiency of the antibiotic to select transfected cells (selection efficiency), as the selection efficiency is based on the difference between the toxic antibiotic concentration for transfected versus un-transfected cells.

Please note that if the concentration needed for selection (as determined by the kill curve), is too close to the toxic concentration for the transfected resistant cells, the selection efficiency will be lower. As a consequence, fewer transfected cells survive and an alternative selection antibiotic should be considered.

To address this need, the investigators in the paper attached below, developed a modified MTT assay of only 3 days to determine the selection efficiency by evaluating the effect of the selection antibiotic on both un-transfected and transfected cells of a given cell line.

The selectivity factor is obtained by dividing the IC50 value of the resistant, transfected cell by the IC50 value of the sensitive, un-transfected cell (IC50R/IC50S). If the selectivity factor is high for a particular cell line, it translates to a high selection capacity for that cell line. On the other hand, a low selectivity factor will result in a low selection capacity for that particular cell line.

For more information, you may want to refer to the paper attached below.

Article Determination of the Selection Capacity of Antibiotics for G...

Regards,

Malcolm Nobre

Transfected cells often exhibit higher sensitivity to antibiotic selection compared to untransfected controls due to several factors:

Possible Solutions:

• Reduce Lipofectamine dosage if cytotoxicity is excessive.
• Allow recovery time before adding puromycin.
• Titrate puromycin concentration to the minimum effective dose.
• Use a lower cell density to improve survival.
• Confirm transfection efficiency using a GFP or fluorescence-based control.

Optimizing these parameters should help reduce excessive cell death in your selection process.