5 replies to this topic

[vzp766]

Hi

We have a problem in our cell lab. When transfecting cells using fugene6, with different plasmids, we see blue dots everywhere (inside and outside the cells) when staining with DAPI.

 

Initially we thought it to be contamination due to the nice blue dots that appear all around the slide. However, we only see these dots when we transfect our cells and not in the untransfected cells.

 

We've already tested a few things:

- we see it in several cell lines using several different plasmids and we just opened a new bottle of fugene6.

- we don't see anything when adding only fugene or only plasmid (only when the two are combined).

- The cells are nice and viable and do not seem affected.

- When incubating either fugene, plasmid or fugene + plasmid in growth media, followed by incubation the weekend over, the media is still nice and clear.

- transfection efficiency is very low

- Results from other experiments where we transfect but don't do IFM (e.g. luciferase, qPCR etc) seem unaffected and look like they normally do.

 

Could it be vesicles with DNA due to poor transfection efficiency? Or some sort of infection which is triggered by fugene-plasmid complexes (sounds odd)? 

 

Please help!! 

Thx

[HawkeyeGrad]

My guess is that you have some sort of infection. Even though you use a new cell line and new Fugene reagents, bacterial and viral infections can linger around on labware, i.e. lab coats, pipettes, incubator door handles, etc and can infect other cell lines very quickly. There may even be some on the caps of the fugene bottle, plasmid preps, or glove box.

 

I would suggest you clean and wipe down everything in your culture room with 70% ethanol and test your cell lines for mycoplasm infection. I would even double check and change the water in your incubator just to make sure. Practice strict aseptic techniques and that may help with the problem.

[bob1]

These bodies could be apoptotic bodies/vesicles that contain DNA

[vzp766]

Thank you!

So, we already cleaned everything in rodalon and ethanol, threw everything (media, PBS, cells etc.) away, decontaminated the incubators. Basically decontaminated everything, and started up from scratch. We also did a mycoplasm test twice and they all came back negative.

 

Together with dapi we also use Acetylated Tubulin antibody and we always see the cells look just fine, so I don't think they are apoptotic vesicles.

 

We did a new test where we mixed fugene6 and plasmid and dapi stained it. What we saw looked exactly like the dots we see. So probably it is due to bad fugene? The fugene-DNA complexes form, but they don't enter the cells. Wouldn't that make sense? 

[bob1]

It is possible, but I would expect the lipospheres containing DNA to be very very small (and not fix or take up DAPI unfixed).  Apoptotic vesicles could come from the cells that die when you transfect using any of these lipid based methods, just because you don't observe apoptotic cells doesn't mean they aren't or haven't been there

[Genelux]

Hi, I hope you are still here.

I think I am having the exact same problem as you described. The DAPI dots only appear after cells are transfected. It is so annoying. Is it really because of the bad lipid? How did you get rid of those nasty dots at last?