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Uses and Applications of FuGENE 6 Transfection Reagent

Troubleshooting Hints if You Observe Cytotoxicity (greater than 5 - 10 % Cell Death)

FuGENE 6 Transfection Reagent has proven to be virtually nontoxic to most cell types. If you observe more than 5 - 10 % cell death in transient transfection experiments, or if selection of permanent transfectants fails, refer to the table below for possible causes.

Possible Cause Recommendation Selection antibiotic added too soon

Repeat transfection and wait an additional 24 to 48 h before adding the selection antibiotic to allow for sufficient expression of the selection marker. Selection antibiotic concentration too high

Repeat transfection using a lower concentration of selection antibiotic. Transfected protein is cytotoxic or is produced at excessively high levels Reduced viability or slow growth rates may be the result of high levels of protein expression, as the cells metabolic resources are directed toward the production of the heterologous protein. The expressed protein may also be toxic to the cell at the level expressed. To analyze cytotoxicity, prepare experimental controls as described below.

Prepare extra wells containing:

  1. Cells that are not transfected.
  2. Cells transfected with DNA alone (i.e., without FuGENE 6 Transfection Reagent)
  3. Cells treated with FuGENE 6 Transfection Reagent alone (no DNA added).

Compare cells transfected with the experimental construct to wells that contain these experimental controls. Consider repeating th 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 r cell density
  • Use different ratios of FuGENE 6 Transfection Reagent to DNA



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