I've been imaging mammalian cells (RPE-1) post-transfection and seen the transfected cells (fluorescent) become static and do not divide, whereas untransfected (non-fluorescent) cells in the same culture do. I've used both Lipofectamine 2000 and 3000 for this and seen the same results for both. I've titred the amount of DNA and lipofectamine, and seen the same no matter how much I decrease it. It is not down to my specific plasmids as I have trialled many different test constructs with different products and observed the same. I observe the cells for five days post-transfection and see no division. So my question is has anyone observed division of confirmed transfected cells? If so, can you recommend reagents or give any advice. Thank you!
Based on your description, it sounds like the transfected cells are experiencing cell cycle arrest or apoptosis due to toxicity from the transfection reagents and/or overexpression of the transfected plasmid.
You can try to:
- Reducing the amount of transfection reagent even further. Start with the manufacturer's recommended minimum and titrate down. Too much lipid can be quite toxic.
- Optimize the ratio of DNA:lipid. Test different ratios to find the sweet spot for your cell type.
- Try a different transfection reagent. Lipofectamine is quite harsh. You could try FuGENE or Polyjet which may be less toxic.
- Reduce the amount of plasmid DNA transfected. Overexpression can trigger apoptosis. Titrate down to find the minimum needed.
- Use a lower CMV or inducible promoter to reduce expression levels.
- Try transfecting plasmid expressing an inert reporter like GFP first to rule out plasmid toxicity.
- Let cells recover overnight in normal media before imaging.
- Check for apoptosis markers like cleaved caspase-3 in transfected cells.
- Monitor proliferation with labels like EdU or by quantifying cell numbers over time.
With some optimization of reagents and DNA amounts, you should be able to find conditions where transfected cells still divide.
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