During differentiation my cells start detaching from the culture dishes. When fresh stocks are thawed and plated die. I am suspecting contamination with mycoplasma. Is my doubt valid? Could there be a possible contamination in my culture? I am going to have them tested for a possible contamination, but if anyone has experienced similar problems please provide your comments.

Very frustrating as one has to wait for a week to complete the differentiation. While when cells die, each time you thaw them amounts for months of wasted eforts. Has anyone has had similar problems? if yes, what solutions were tried? Many thanks

Aspeedasai

Hi! The easiest way to check for mycoplasma is to just plate cells on cover slips for a day or two, fix with methanol or PFA, permeabilize and stain with DAPI. Mycoplasma can be easily visualized as small blue dots clustered a little way away from the large blue eukaryotic nuclei.
If you do have mycoplasma contamination, just look over this forum for info on reagents you can use to clear them, and clean up your environs.
-sam

Where are the cells from? I have been hearing this complaint a lot recently. I have heard of the cells dying off during expansion or differentiation. Are your cells ok during expansion? Most of the complaints I am hearing have come from researchers who recently acquired cells from ATCC.

Hi, thanks for your suggestions. The cells have been quite happy in the past, and worked quite well in my hands. I have done everything on them including retroviral infection for gene overexpression, differentiation and what not?

The problem has suddenly cropped up, although it will take couple of days time to detect the infection. I am almost sure that it is not due to the culture conditions. As I carried them to a separate facilty, and saw the same mortality, upon thawing a fresh vial from liquid nitrogen. I hope that the infection has not spread to the cells others had in the incubator, Ooops.....

Has anyone experienced with 3T3-L1 cells ever encountered mycoplasma infection? If yes, did that kill your cells in culture? Utterly frustrating this, I see that it will cost me atleast 2 months of wasted time, to rescue the cells or start from scratch with fresh cells from commercial sources. I wish I could get as tiny as them to Kick some a;;e ;-(

If the cells are not contaminated, amybe you want to look at the FBS your using. Has it changed? We've had some wierd things happenwith FBS. Cells can grwo great for several passages and then suddenly hit the wall and stop growing. Lots of testing led us to the FBS.

Could anyone please comment here if, there is a contamination affecting adherance properties and lead to cell death?

Certainly, I do not have changes in the serum batch.

Aspeed

Hi! In a desperate effort to find out what happen in my 3T3-L1 culture I found this forum. I have the exact same problem on my cells. Just from the day 2 post-differentiation the cells start detaching from the dishes, specifically from the sides of the well. Then, on the next days the "hole" evolves, and the cells end up dying. This turn up really desperating because I have tried several things by now: I clean up and disinfected the incubator, replaced all reactives, tried different FBS lots, different serum concentration (5% and 10%), different plates (6w, 24w, 96w...costar and nunc), different cell concentration for differentiation (not confluent and very confluent)...and nothing seems to work. I have serious thoughts that the problem maybe a contamination because the cells worked fine in the past, so I`m going to test for mycoplasma, although I have the same question: Does anyone knows if a mycoplasma contamination in this kind of cells results in this conditions?. Another interesting thing that I noticed is a weird medium consistency, like oil, every time that I changed the media from the cells which already have these holes. When I`ll have some results it will be posted here, meanwhile if anybody knows something, I`ll be waiting
Thanks!, and good luck - DGARDIAZ

Try to do the DAPI staining and look for the lttle dots that spell Mycoplasma - though the pCR is required to be really sure. Contamination can lead to cell death - which can be induced by LPS for instance. Let us know whats happening - lots of people seem to have these issues.

Hi Friends,

Certainly people are doing everything else except testing them for mycoplasma. So, I will have the results for the test next week. If the test is positive, I am going to be harsh on these mycos with some flouroquinolones. Recovering them will take two weeks, and I hope this will not affect the stability of overexpression (gene I introduced them using retrovirus).

Lets see.... Gardiaz, its strange you tried everything else except testing them for mycoplasma. Are you considering a test soon? Pls prompt me on any progress you have on the situation. The nature of problem we have is exactly the same.

Good luck
Aspee

Hi
I wonder if you find a solution to the 3T3-L1 detachment peoblem.
Kivanc Birsoy
Rockefeller Universsity
kbirsoy@rockefeller.edu

Hello,

I am not sure if you are trying and transfection/ retroviral infection protocol on your cells? I checked everything including mycoplasma contamination. My cells were negative.

In conclusion, the cells are neither infected nor your media, plates affect them.

The problem is if your reagents including any viral material or treatments may be too harsh on the cells, they loose the adipocyte phenotype and revert to being pure fibroblasts.

So the solution is to change your cells and obtain a fresh vial where people are happy culturing them. PLEASE! Modify your treatment conditions. For example, I used lower retro-viral load and took care not to allow cells to become confluent (100%) anytime. I am happy that I have resolved the problem.

Hope this helps

Good luck

Aspee

I have same problems, too.My cells are detaching from the wells.I understood that differentiating cells before they become fully confluent.Is that the solution?

Hi all,
I've just posted a similar topic just because I didn't understand which is the solution that Aspeedasai found....
Can you explain me?
Thank you
Silvia

Hi all,

I am having issues with 3T3L1 differentiation for the past one month. I have been using cells from ATCC and they worked fine in my hand earlier. Its just for about a month now, that when I add my differentiating solution (IBMX + DEX in DMEM with 10 % fetal bovine serum) I see little round dots cluttered together floating over my cells exactly two days after treatment.I seed my cells at 100% confluency and two days after treatment I see them about 20 - 30 % confluent with a clutter of small dots floating over. It doesnt look like bacteria and there is no color change of medium (you know how the medium becomes yellow if its used up by bacteria or cells) which makes me think maybe its cell debris thats floating.

However I have been using the same protocl same concentrations as before. The solutions I make ( IBMX + Dex in DMEM + FBS) look fine without cells, but when I add it to my cells its looks contaminated.I also thawed different vials
(though subcultured from the same stock) and the probelm seems persistent.

I am not sure if its contamination or my cells are just dying. I am using the IBMX and DEX provided by Chemicon (chemicon adipogenesis kit).

I would really appreciate any help. This has been very frustrating.Looking forward to hearing soon.

Rgds,
Priti

Hi priti
You should definitely check for mycoplasma using the basic DAPI protocol mentioned earlier in this forum /post series, a PCR check may also be required. If all your vials exhibit the same problem, the original stock might have been contaminated. Removing mycoplasma takes about 3-4 weeks of continuous culture with MRA, normocin or similar compounds.
Also, you mentioned that you plate your cells at 100%: why do you do that, and how do you do that (i.e., do you just lift off a plateful of cells, and transfer tthe entire contents to another plate?!)?
-sam