3T3-L1 Differentiation Media Preparation Help - (Apr/03/2007 )

Is there anyone who can help me on how to prepare a differentiation media for 3T3-L1 cells in detail?

There is a lot of values for insulin, IBMX and DEX.But how I can prepare the stock solutions of these?Also can I stock the prepared differentiation media or do i have to use when i prepared?
In which solutions do i have to dissolve the IBMX,DEX and Insulin.

I yet started to work with 3T3-L1 cells.(actually my first animal cell).I just want to see differantiating.

Thanks for your interest.
Regardly...

Hi Lazarus,

I must agree with you, there are plenty of different concentrations at witch you can induce 3T3-L1 to differentiate into mature adipocyte. In our lab, we induce them with 0.5 mM IBMX, 1uM Dex and 5 µg/ml insulin. The dexamethasone is sold either in a water soluble powder, or a non-water soluble form. Be careful to correctly calculate the final concentration if you work with a water-soluble form The IBMX is soluble in DMSO, and the insulin we use is already in solution (Sigma).

I suggest you make your differentiation media fresh every time.

Hope this helps!

Thanks for help.I am trying now and waiting for differentiation.Hope i will see droplets in 4 days.Thanks again.

Another problem was occured during differentiation.My cells start to detach from the wells at 3rd and 4th days of differentiation.Any idea why and what can be done?

This seems to happend when your cells have reached confluence once before the differentiation. Have your cells become confluent during your passages? If so, you might want to start with fresh cells and make sure they never reach confluence.

It may be due to a high number of passages too. Make sure your cells are at low passage.

I hope you meant 0.5 uM IBMX

Hi Lazarus,

I must agree with you, there are plenty of different concentrations at witch you can induce 3T3-L1 to differentiate into mature adipocyte. In our lab, we induce them with 0.5 mM IBMX, 1uM Dex and 5 µg/ml insulin. The dexamethasone is sold either in a water soluble powder, or a non-water soluble form. Be careful to correctly calculate the final concentration if you work with a water-soluble form The IBMX is soluble in DMSO, and the insulin we use is already in solution (Sigma).

I suggest you make your differentiation media fresh every time.

Hope this helps!

No, I really meant millimolar (mM).


Why?

Because we use this following differentiation cocktail:

Cell medium (DMEM) + ten percent of FBS containing 1 uM (i.e. micromolar) Dexamethasone and 0.5 uM IBMX + insulin (10 ug/ml)
Have you got any reference about it...I m gonna search some...
Keep in touch
Silvia

ok I ve just check my protocols and we use to differentiate as I told you 1 microM DEX and 0.5 microM IBMX and our cells easily differentiate even if you re right and data from literature say that it should be 1 microM DEX and 0.5 milliM IBMX. So we create a new protocol, that works well!!

Bye!
Silvia

Actually all of my references gives the protocol with 0.5mM IBMX.Generally they differ in DEX and Insulin conc.