Hi Farah Diab, the shape of your cells after adding the mix looks fine. IBMX can be very stressful for the cells so if you look at them few hours after adding you differentiation starting mix, you'll always see something.

If your cells detached, it's probably because there is a concentration problem in the mix you are adding or that change the pH.

How are resuspending your IBMX? in DMSO? What is the dilution you are using in your media?

Hi Cécilia Colson and thanks for your answer!

Actually, as you already said IBMX is a stressing compound for the cells, this was regularly monitored throughout different differentiations!

However, yes I usually resuspend it in DMOS and the used DF= 1.6 (for example for 16mL of media, I add 9.6µL of IBMX).

Hi Farah Diab and Cécilia Colson

I also experienced this problem. I used 0.5 mM ibmx by preparing it in methanol.

Could this problem be caused by solvent or high concentration?

What should be the storage conditions for ibmx

thank you

Hi Farah Diab,

First, sorry for the delay but I did not have the notification of your answer. I hope you found a solution to your problem.

If not, what is the final concentration of IBMX in your culture media? I was using 0.5mM on human pre adipocytes but it could be too low for 3T3-L1. Depending also on your stock concentration because DMSO should not be in high amount in your culture media. IBMX can be very tricky to prepare.amounts

Also, are you adding your media in the center of your well or on the side, slipping along the wall?

Hi Ezgi Nur Çil ,

IBMX can be stored at -20°C if it's in solution with a solvent. I don't know if methanol is a good call to add on the cells, it's very toxic. I would suggest to using DMSO instead. Your final concentration is fine, 0.5mM, but you stock solution should be at least 1,000X.

Hope that helps