3T3-L1 curling

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snowday
snowday's picture
3T3-L1 curling

The curling of 3T3-L1 seems to be a common problem, and after reading many solutions, I still am encountering difficulties. 
Here is the problem. My cells proliferate well until changed to differentiation medium.  This is where the problem happens.  The medium is replaced with short-term differentiation medium, containing DMX, IBMX, 10% FBS and insuline from bovine pancreas, which has been heated to 37 C and pH adjusted to 7.2. After 2 days in the medium, the cells appear fine, no curling has happened, but the medium has now turned yellow! When changing the media to long term differentiation, containing only FBS and insuline, I can litteraly see the cells curling before my eyes!

Here is what I have tried to remediate the problem:
-Warmed the medium to 37 before using.
- Used a 1ml pipette instead of succession so as to not disturb the cells during the change.
- Added the long term medium slowly with the pipette pressed against the edge of the well wall at 90 degree angle (or almost)
- With an initial 0.5 ml in each well (24 well plates) I remove 375 ul and add 475 ul (compensating for evaporation)

I still get curling! 

Any ideas or solution?

Chuck Miller
Chuck Miller's picture
 It sounds like you may be

 It sounds like you may be letting the cells overgrow. Are the cells post-confluent? Can you try lower cell densities? 

Chin Fen Teo
Chin Fen Teo's picture
 Hi snowday,

 Hi snowday,

I can't quite picture the "curling" shape that you are getting in my head, Is it possible for you to upload an image? Differentiated L1 will undergo phenotypic change starting from Day2 indeed, as the fat vacuoles start forming in the cytosol.

The yellow color in your medium doesn't sound right. I never encountered such problem before. Do you have Pen/Strep in your medium? And how do you prepare the differentiation medium? 

In the protocol that I used,  L1 preadipocytes have to be 100% confluence (for 2 days) before feeding them with differentiation media, hence I don't think cell density is the culprit for your problem...

Cheers.

wwwong83
wwwong83's picture
 Hi,

 Hi,

I'm a new user here. Actually I had try to do the adipogenesis on 3T3-L1 for past few months, but it ends up with the curling problem. May i know that what's the perfect protocol for the adipogenesis?

My protocol is:
Day 0: Seed 20,000 cell at 6 wells plate
Day 2: Add 0.5mM IBMX and 1uM DEX in DMEM (with 10%FBS and antibody) + drug treatment 
Day 4: Add 10ug/ml insulin in  DMEM (with 10%FBS and antibody) + drug treatment
Day 6: Drug treatment
Day 8: Drug treatment
Day 10: Oil Red o Staining

Another problem of this experiment that i faced is during the oil red o staining, the llipid in the cell cannot be stained. Do anyone of you face this problem?

I'm getting crazy after running this experiment as it ends up fail.... My supervisor keep on asking how's the result.....

Best Regards,
Weng Wai

snowday
snowday's picture
Hi Pippuri and Chuck,

Hi Pippuri and Chuck,

My curling problem has subdued itself quite a fair bit but I am now stuck with another major problem. When I perform the adipogenisis assay on the 7th day of differentiation, levels of lipid accumulation for the positive control (Rosiglitazone) is very low. In fact, it is almost as low as the vehicle (which is for its part slightly high). Interestingly, when I look through the microscope, the positive control seems to have more lipids then the vehicle (DMSO).. but the absorbance measurements say something different.

Has that ever happened to anyone?

As for the colour, I am still having that problem. I was explained that the cells excretions may be acidifying the medium. I do have Pen/Strep in my medium. And the differentiation medium is prepared like so (the pH is subsequently adjusted to 7.2)
Short term (for 100ml):
Medium (DMEM, 10% FBS. 0,5% P-S)
IBMX (250 uM)
DMX (1 uM)
Insuline (500 nM)

Long term (for 100ml):
Medium (DMEM, 10% FBS. 0,5% P-S)
Insuline (500 nM)

I also have a hard time understand how overgrowing the cells can be a problem. As far as I am concerned. The cells have to be fully confluent to arrest proliferation and initiate differentiation.
So like you Pippuri, I wait 2 days before differentiating..

Any advice anyone??

Thanks.

Michel