Can anyone help me to identify the differentiated osteoblast MCT3T3-e1 cells from pre-osteoblast cells after treamt with 25 µg + 10 µM β- Glycero Phosphate + 10nM Dexamethasone.
1. How we can identify the cells were differentiated?
2. Whats is the approximate time incubation to visualize the prominent differentiated cells or bone nodule formation in it?
3. How many days after we can check for gene expression by RTPCR?
Please suggest me trouble shooting steps to know about these work in deatil.
Hi Karthi,
For standard differentiation of your osteoblastic cell line with Glycerophosphate, Dexamethasone and Vitamin C, you can regularly check under the microscope, when the cells start to deposit calcium and you will get a mineralized matrix, you will see a black cloud on the cells indicating mineralization. This normally can take up to 21 days with twice medium change a week. Sometimes, it even can take 28 days, for example in osteoprogenitor cells like bone marrow-derived stromal cells. Normally after two weeks you will start to see some calcified regions in the wells. For RT-PCR, you can check after 3-7 days. This process is quite fast.
For troubleshooting, you might not see any minerlaization, then you should refresh your differentiation medium and use alpha-MEM containing 10% FBS, P/S, 100 nM dexamethasone, 10 mM β-glycerophosphate and 0.05 mM L-ascorbic acid-2-phosphate. You can purchase them from Sigma-Aldrich. Good luck.
Hi Adel Tekari
Thank you for you suggestion and i will optimize my study according to your protocol..
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