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Help with HUVEC cell transfection

HUVEC Transfection

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3 replies to this topic

#1 Bonjo7

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Posted 28 May 2015 - 06:11 AM

I am transfecting HUVEC cells with wild-type or point mutated forms of a gene that is cloned into pcDNA3.1. Post transfection, my cells are losing their adherence and, though I am not sure, I suspect that a significant proportion are dying.

 

I am using a jetPRIME transfection reagent designed for HUVEC cells according to suggested protocol.

 

This issue occurs when I use either transgene or vector alone, but not when I perform a mock transfection where no DNA is added.

 

The DNA is phenol/chloroform extracted so should be free of contaminates, so I think that the DNA itself may be harming the cells (I plan to do another phenol:chloroform extraction to be sure, though.

 

The plates are coated with 0.2% gelatin.

 

Not a lot of supervision here so I would very much appreciate any assistance.

 

Thank you!



#2 TheFOrsh

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Posted 29 May 2015 - 12:17 PM

HUVEC can be a pain to transfect and many endo/epithelial cell types show low viability when transfected with DNA.  When I first started I found siRNA transfection wasn't too bad (maybe 20% death compared to 10% I was getting with non-endo cells) but plasmid transfection had very low viability, most cells would just die.

 

I've not actually worked with HUVEC, but from the endothelial lines I have worked with I can recommend a few things that helped me.  I also used jetPRIME, though I found transIT-LT1 to be slightly better in preliminary trials I finished up the work before I got a chance to test it further.

 

First, transfect when cells are still in exponential growth phase if you can.  Second, replace the media with fresh complete media 4-24 hours after transfection - you might want to try a range of times to try and optimise the experiment (4, 8, 18, and 24 hours is a good start).  Third (and probably most important!), reduce the amount of DNA you're transfecting with - I went with 400, 800, 1200 ng of plasmid, and used 0.5, 0.5, and 1.0 uL of JP with that respectively, though you could also try titreing the JP conc.  I found when transfecting endo cells 'less is more', often the lower concs would have a higher viability and also better expression of my plasmid.

 

 

Good luck



#3 TheFOrsh

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Posted 30 May 2015 - 04:43 AM

I should add - my transfections were in 6-well plates.  Adjust your quantities accordingly.



#4 Bonjo7

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Posted 31 May 2015 - 05:01 PM

Thank you.

 

It does seem likely that plasmid DNA is reducing viability.  I will follow this advice and try using less. 






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