Hi,
I am using C3H10 and MC3T3 cells both from mouse origin. Im using trans gene 9 transfection reagent for transfection. But the efficiency is very low. I am using 600ng DNA / well for 24 well plate. Can someone suggest any modifications?
Thanks,
Jaya
I haven't worked with the cell lines you listed, but given that they're more or less of a mesenchymal/osteoblastic origin, you might try using a transfection reagent initially designed for NIH-3T3 cells (https://altogen.com/product/nih3t3-transfection-reagent-swiss-mouse-fibroblasts/). They're mouse fibroblasts, and although transfection efficiency will be reduced, it should still give you better results.
Hello,
I have not worked with these cell lines, but if transfection efficiency is low, I tend to try the following:
1. test a (wider) range of different ratios of transfection reagent and plasmid / DNA concentrations
2. try a different transfection reagent (Lipofectamin and Polyfect work well in my experience, calcium phosphate might be a cheap and effective alternative)
3. If my target construct is rather large, I tend to try smaller control vectors to exclude that vector size is an issue.
4. Some people use chloroquin to enhance transfection efficiency, but that hasn't helped me in the past. But it might be worth a try.
5. Although it shouldn't be an issue, but is sometimes missed for exactly that reason, you should (re-)check that your promoter is working in the cells you are using. If those cells are commonly used by other researchers as documented by the literature, check whether they do anything different and try their protocols.
6. Check for mycoplasms in your cells. Contamination with those little beasty things can really decrease transfection and transduction efficiency.
Furthermore, if your low transfection rate is not only in control cells, it might be possible that there is a biological effect. The expressed construct might be targeted for degradation by intracellular pathways, which you did not know of beforehand. In that case, you could check for mRNA levels. Sometimes, construct effects might also cause cells to throw out the plasmid again.
If your lab is equipped for that, you might also want to consider viral transduction instead of transfection. Usually, this is quite efficient.
Finally and if nothing helps, whether you really need a high transfection efficiency might also depend on the experiments to be used. For subsequent Western blotting, higher transfection rates are often needed to clearly make out biological effects. However, immunocytochemistry, for instance, also allows you to work with lower transfection efficiencies as long as you are capable of identifying transfected cells (e. g. by having flourescence tags). If you really need a specific cell line, which does not work well in transfection, you might consider using ICC instead of Western blotting, although there are, of course, some disadvantages to that.
That would be my standard strain of thought in such a situation.
Cheers and good luck,
Jan.
Ialso did not worked with these cell lines, but 600ng/well for 24 well plates shounds very high to me...( I have used 250 ng/well for 12 well plates)
Try to make transfection with several concentrations - I think that will do the trick
other transfection reagents that you could use (in addition to Jan's suggestions) are siPORT NeoFix, HighPerfect, or much cheaper and also quite effective: PEI
First, check your DNA integrity and actual concentration.
Make sure your transfection reagent is okay.
Test these two things in standard condition.
I use Xfect as it can be used with serum and makes transfection easier. It is also pretty cheap. Also, try transfecting with a simple GFP vector to see if it is the plasmid you are using or the transfection reagent. If they all glow green with the GFP vector, then your construct has a problem. If they are not green with the GFP vector, then try different transfection reagents or ratios of DNA:reagent or electroporation.
Good luck!
When you have low efficiency with lipofection or polyfection based reagents you can try a new magnetic nanopaticles based method called Magnetofection that has great efficiency on primary and hard to transfect cells
http://www.ozbiosciences.com/content/13-magnetofection
This method has been sucessful for transfectiong siRNA in MC3T3 cells
http://www.ncbi.nlm.nih.gov/pubmed/20937352
If you need any further information let me know
I haven't worked with the cell lines you listed, but given that they're more or less of a mesenchymal/osteoblastic origin, you might try using a transfection reagent initially designed for NIH-3T3 cells (https://altogen.com/product/nih3t3-transfection-reagent-swiss-mouse-fibroblasts/). They're mouse fibroblasts, and although transfection efficiency will be reduced, it should still give you better results.
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