Please Help! Transfection: dropwise addition of lipid -dna complexes - (Dec/05/2009 )

When I do dropwise addition of lipofectamine-dna complexes(in OPTIMEM) in 6 well plates(293 cells), I find it very hard to mix the drops with the DME serum media that I use for the cells. Sometimes I tilt plate forward then tilt back after every few drops. Other times, I try to keep the plate flat and drop while swirling the plate in a circular motion. Nearly every time I transfect, I look under microscope after adding the complexes and notice that large ammounts of floating cells appear, many more floaters in some wells than in others. Am I doing something wrong?

I think I may be trying to hard to mix the optimem+complexes with the serum media the cells are in, and that the force(although I try to be gentle) resulting from my thoroughness is enough to cause cells to clump and detatch from plate.

I am concerned because my plates were ~70% confluent this morning, but after I changed media and added the lipid-dna complexes, it seems like only 50%(or fewer) cells are attached. Is there anything I can do to fix this before I harvest tomorrow morning?

Please advise.

Thanks in advance.

Today I looked at the cells and they seem to have clumped up in some parts of the plate. In these parts they are very very dense. On other parts of the plate they look a little more spread out. Would you recommend that I trypsinize these cells so they can spread out over the surface of the well?

try "reverse transfection" - transfect as you seed out your cells. Also saves you a lot of time.

Thanks Bob, I was reading about reverse trasfection earlier today. An invitrogen guide mentioning Lipofectamine 2000 suggests using reverse transfectin for stealth Rnai or Rnai. I am transfecting plasmid DNA. Does reverse transfection still work?

Also, is the nucleic acid in the media responsible for cell-cell clumping? If so, how could I prevent it from happening when doing a Reverse transfection.

Thanks for the reply

I use reverse tx for all my transfections including plasmid; it works fine. In fact it should work better as the cells are round and unattached, so more area is exposed to the complexes.

The clumping isn't because of the DNA of the transfection reagent, It is much more likely that you over trypsinised the cells or that there is a vibration in the incubator which will cause the cells to settle in concentric rings (in dishes /6 well plates - you won't be able to detect it in 24 well plates) or lines in flasks. This can be intermittent and is usually more obvious with cells that are taking a while to settle (like yours) as they have more time to get shuffled into the waveforms.

Also don't believe the optimem stuff invitrogen goes on about. Just use your normal medium (without FBS), it will be fine. I have had less toxicity with Fugene 6 in the past too, if that is any help.

Thanks for the suggestions Bob. I will take these into account as I continue these experiments.