We are trying a protocol for differentiation of 3t3 cells that involves exposing them to insulin, dexamethazone and IBMX with poor success. Any further ideas?
Heidi,
Since you were reaching near 100% conversion, it is a matter of troubleshooting, not necessarily looking for a change in protocol. You have to think about what has changed since then... besides the passage number of your 3T3. High passage number cells often start behaving differently and the simple solution is to go back to your frozen stock.
Heidi ,
I am using 3T3-L1 cell line , for the differenciation , I seed the cells until confluence. After 48 hours I add the mixture for differenciation for another 48hours which contains :
0.5mM IBMX
2.5mM Dexamethasone
10µg/mL insulin
I change the media to use a maintenance media containing 10µg/mL insulin , and I reach 90% of differenciation 9 days post-MDI .
ps : I use DMEM 4.5g/L glucose
Hope it helps
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