I did lipofectamine 3000 tranfection in neuronal cell line. I used 2 ug of pmaxGFP. After the DNA-lipid complex was formed, it was added into cell for 3 h. Then transfected media was removed and replaced by conditioning media which induce morphological alteration.
I observed the transfection efficiency after 24 h. I found that most cell (80% ) were expressed GFP fluorescence very well. The problem I saw was the toxicity of strong GFP-transfected cells and these cells cannot undergo morphological change, while un-transfected cells can normally express the induced morphology as usual. It seemed that highly GFP-expressed cells annoyed the morphology alteration.
How was the problem ? Is it because I used high concentration of pmaxGFP?
Next time I have plane to used 0.2 ug GFP instead but will keep all protocol the same.
Your result is kind of weird. Usually I use 2μg plasmid and the transfection efficiency is about 50%. You can try 0.2μg and you can also try to change the time point to 12h. Maybe the toxicity is too high and the cells are died after 24h. So they can not change the morphology.
you could set some control groups:
1: mock transfection: no plasmid, lipo
2: empty plasmid transfection: no protein expression plasmid, lipo
3: EGFP transfection
if all of these groups see the "annoyed the morphology alteration", you would need to change the transfection reagent.
if 2 and 3 groups see the "annoyed the morphology alteration", that means that you would need to get higher quality plasmid (purify, endotoxin removal, etc.).
if only 3 see the "annoyed the morphology alteration", use other protein for marker such as RFP, etc.
Xin Ren Yes, that is what I have planed to do next. I will 10X dilute the plasmid concentration to 0.2 ug first. If it doesn't work well I will reduce time to change. Thank you for discussing.
Do not transfect too much DNA and always follow the rules of Invitrogen lipofectamine guidelines. It may be possible that you add too much lipofectamine in the reaction which is the main cause of toxicity. Standardize lipofectamine : DNA ratio. I think you have used 6 well plates. Also if the cells are too sensitive to lipofectamine you can try other transfection protocols also.
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