Help! Clontech Xfect is killing my T-Rex HeLa cells!!! Why! - (Jul/22/2012 )

Hi all,

I am working with T-Rex HeLa cells from Invitrogen. I was transfecting them with Clontech's Xfect and then, all of a sudden, the Xfect started killing 90% of my cells. I'm having just as much trouble with Lipofectamine. GeneJuice seems to be ok.

I'm plating my cells on acid washed fibronectin coated glass slides. The cells are perfectly happy, then within an hour of adding the DNA+Xfect mixture, I see massive cell death. Xfect is supposed to be fine for HeLas according to the Clontech website. I'm adding the recommended amount of Xfect and 5ug of DNA/transfection (I'm doing co-transfections so a total of 10ug of DNA). A couple of months ago I was doing the same experiments with the same cell line and all was well.

I've changed out my media, the fibronectin, and my Xfect reagent. Nothing helps.

Has anybody else encountered this? What's going on? I can switch to FuGene or GeneJuice but it's expensive to just waste the Xfect. And I have no idea why this is happening!

Help!

Thanks y'all!

Cheers!

Are you sure they are dead? Lifting off is not necessarily a sign of death, they may well re-attach with further incubation.

10 ug (or even 5 ug) of plasmid is a massive amount for the amount of cells you can seed on a slide - I use 0.25-1.0 ug per well of a 6 well plate and that is heaps. Perhaps this amount of plasmid is killing off the cells.