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Transfection problem


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4 replies to this topic

#1 jaya2020

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Posted 22 September 2014 - 08:01 AM

Im using Xtreme 9 transfection reagent for preosteoblast cell line. But almost 75% of my cells die within 24 hrs. Why is this so. Is there any toxicity with the transfection reagent.



#2 rkay447

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Posted 22 September 2014 - 12:15 PM

Yes, all transfection reagents have some toxicity issues and you are going to need to optimize.  First, different reagents have different efficiencies and toxicities.  There are reagents specifically made for "hard to transfect" or "toxicity sensitive" cells.  Next, how are you prepping your DNA?  Are you using an endotoxin-free kit?  Again, some cells are more sensitive to the endotoxins that come out of bacteria and are often in DNA preps unless you are using a kit specifically designed to remove these contaminants.  Finally, are you using antibiotics on the cells?  Even if you are using antibiotic-free serum during transfections, I've seen some cells that are simply hypersensitive and if you culture them with antibiotics the day before or after, they die.  



#3 jaya2020

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Posted 22 September 2014 - 01:20 PM

thanks for the reply. I dont know about the endotoxins because the DNA was prepared by someone long time ago. Im using antibiotic serum free for transfection,  Can you tell me which company provides the kit for these type of cells, which are hard to transfect



#4 rkay447

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Posted 23 September 2014 - 08:47 AM

The first thing I would do is reprep the DNA using an endotoxin-free kit.  You'll have to retransform the DNA back into e.coli, pick a colony and do a plasmid prep.  DNA quality is a critical aspect of successful transfections.

 

Then I would try transfecting using lipofectamine LTX with plus reagent or the new lipofectamine3000.  This is what has given me the best results but you can also try x-tremeGENE from Roche, or Fugene from Promega.  You can usually call and request a sample.  That way you can test a few different reagents and see what works best for your particular cell line.  

 

Finally, do you wash off the transfection about 6 hours afterward?  Even if the directions say you don't have to do this, I found it increased survival tremendously.  About 6 hours after the transfection, wash gently in PBS and give fresh media without antibiotics.



#5 jaya2020

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Posted 24 September 2014 - 06:13 AM

Thank you. Iam using the kit from Qiagen for DNA preparation. As you suggested, Iam going to try different transfection reagents. Till now I have not changed the medium after 6 hrs. I will try this too next time.






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