6 replies to this topic

[gantzgraf]

Hi, I would be very grateful if anyone can suggest anything to help me here.

I have been transfecting HEK293 cells successfully with a GFP fusion protein using Lipofectamine 2000. I optimised the transfection as suggested by the manufacturer and found them to be successful at all ranges tested (Lipofectamine:DNA ratios of between 1:0.5 and 1:5). The optimum happened to be around the manufacturer's suggestion of 4 ug DNA and 10 ul lipofectamine in a well of a 6-well plates but in all cases there was acceptable transfection efficiency and no noticeable cell death 48 hours post-transfection.

I've then performed further transfections with which to select stable cell lines with no appreciable cell death until i started performing selection.

A few (maybe 5 or 6) passages later I've been trying to repeat my transfection under the optimised conditions in order to perform some experiments but when i come to look at the transfections the next day they're dying. By 48 hours almost all cells are floating. This is true when performing the transfection in flasks or on poly-d-lysine coated coverslips in 6-well plates.

My colleague has found the same thing - she has been passaging the cells independently, transfecting with a plasmid encoding a different protein and using a different tube of lipofectamine. While her transfections worked initially she now also observes cell death 24 hrs post transfection.

In both cases there is no sign of contamination even after leaving the flasks/plates in the incubator for a further week or so.

So, we don't believe the DNA and the lipofectamine are the problem as both have performed fine previously and we''re sure we're not getting infections. We've even tested the FCS we're using because we have recently changed supplier. We are at a loss and can only think of thawing another vial and seeing if those cells will transfect ok.

Can anyone think of any possible reasons for this? My colleague and I are getting very stressed out over this holding up our work...

Thanks for reading,

Dave

Edited by gantzgraf, 29 July 2009 - 02:36 AM.

[Superman]

I am not a pro I must admit but I have been doing some transfections of late. What I was told was that 4ug quite a high amount of plasmid DNA in 10ul of lipofectamine. My transfections worked great with 2ug in 10. I heard that the higher the DNA the more chance they have of dying so perhaps try to use less DNA and see what happens.

Don't know if that will help but it's all I have im afraid

[gantzgraf]

Superman, on Jul 29 2009, 12:07 PM, said:

I am not a pro I must admit but I have been doing some transfections of late. What I was told was that 4ug quite a high amount of plasmid DNA in 10ul of lipofectamine. My transfections worked great with 2ug in 10. I heard that the higher the DNA the more chance they have of dying so perhaps try to use less DNA and see what happens.

Don't know if that will help but it's all I have im afraid

Thanks for the reply. I take your point, but it has worked fine with 4 ug in the past and it also happens to be the amount recommended in the manufacturer's protocol for this kind of culture vessel (so 4 ug DNA in 250 ul Opti-MEM added to 10 ul Lipofectamine 2000 in 250 ul Opti-MEM, incubate for 20 mins and add to cells growing in 2 mls medium). So it does seem odd that all of a sudden for 2 people working independently that the same amount of DNA would suddenly become cytotoxic (we are both using the same maxi preps as for previous experiments also).

I didn't want to write for too long in the original post but I did try reducing the amounts of DNA to no avail. I guess the next step is probably to thoroughly re-optimise the whole procedure but I'm concerned that there is a more fundamental problem I have yet to consider.

[genehunter]

Was transfection done with high cell density?

[gantzgraf]

genehunter, on Jul 29 2009, 04:26 PM, said:

Was transfection done with high cell density?

Yes, I aim for 95% confluent. However, my colleague tells me that before things started going wrong her transfections were still fine when her cells were nearer 70-80% confluent. Regardless we have tried to make sure that cells were at least 90% confluent since things have been failing.

[scolix]

If the cells have gone through a certain number of passages, then they start to die after 48 hrs. Try to get new cells and repeat transfection.

[yoohoo]

I have been using Lipofectamine 2000 and Lipofectamine LTX myself. How long do you let your Lipofectamine stay out at room temperature? The liposomes may degrade over time There is a simple method of fixing this by using an ice block. Ensure that the tube doesn't remain out for too long.