In fact, you will find that differentiation cocktail varies from one paper to another. So, as long as your cells differentiate, you're fine

AAAAAAAAAHHHHHHHH

48 hrs after differentiation of 3T3-L1 with dexamethasone 1 uM, insulin and IBMX 0.5 mM the situation is DEVASTATING
All cells are dead and detached from the well....Any idea about the citotoxicity of IBMX

Thank you, I m so frustrated....

Silvia

Have your cells reached confluence before differentiation? If so, as I have experienced a lot of time, cells tend to float as a whole sheet, and do not seem dead.. but don't differentiate. Maybe, if your passage number is too high, your cells won't differenciate neither.

I checked again rapidly on pubmed, and the first three publications I checked were using 0,5 mM IBMX.

As for the toxicity, the IBMX is only soluble in DMSO. But in the concentrations we are working with, I don't think the DMSO is toxic. And the biological effect of IBMX is to inhibit the c-AMP phospho-diesterase activity, witch in turn has the effect of raising intracellulare c-AMP concentrations... and this is not toxic.

So in my opinion, either your cells have reached confluence before being plated for differentiation, or either they are too old.

Neither one or the other could be the possible explanation: we let cells reach confluence one time in their life, that is just before adding the differentiation cocktail. We NEVER let them to confluence if we have in mind to split them again after...Even if this means to work on sundays!!!

Moreover we thaw these last cells that were a P1 and differentiated them at their P3... You agree with me that it s impossible to do better!!
Of course DMSO was 0.001 per cent (i.e. 1 per mill) in culture medium so well below worrying concentrations!!
Keep in touch!!
Silvia

Hmmm

I really don't see what could explain this then.. These cells seems to "have to be in the mood" in order to differentiate

But anyway, if you have always worked with 0,5 uM of IBMX and have the mature adipocyte phenotype with all the genes and transcription factors expressed, well, stick with your protocol! Or try with new cells, it may work also!