e experiment with a secreted reporter gene assay such as SEAP, hGH, or a standard -gal control vector. Cells secreting SEAP should show little to no evidence of cytotoxicity.

The culture may be contaminated with mycoplasma

Use the Mycoplasma Detection Kit (Cat. No. 1 296 744) or Mycoplasma PCR ELISA (Cat. No. 1 663 925) to determine if the culture is contaminated. Treat the cells with BM Cyclin (Cat. No. 799 050) to eliminate the mycoplasma. Alternatively, start the transfections over with a fresh uncontaminated culture.

Cells may not be healthy (e.g., malfunctioning incubator, media problems)

Assess the physiological state of the cells and incubation conditions (e.g., CO₂ and temperature levels). Perform the same controls as suggested above for cytotoxicity to eliminate the influence of transfection reagent or nucleic acid. Plasmid preparation
contaminated with large amounts of endotoxin

Endotoxin is reported to be cytotoxic to some very sensitive cell lines (e.g., Huh-7) and primary cultures. By using FuGENE 6 Transfection Reagent for many common cell types, it may be possible to use DNA containing higher endotoxin levels. If above tests prove negative, FuGENE 6 Transfection Reagent may be cytotoxic to your specific cell type If you are using a very sensitive cell line, measures can be taken to minimize cytotoxicity:

• Perform the transfection in the presence of FBS
• Reduce the time of exposure to the transfection reagent:DNA complex to 23 h, then replace the medium
• Perform the transfection at a highe
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