Hi Mahsa. What kind of plasticware did you use for culture, and at what density were the cells seeded at? Did you induce differentiation at or before 100% confluence?

I think the culture aged. What day of growth was photographed? According to most methods, staining must be carried out on the 21st day, however, the culture often does not live up to this time. Therefore, it is necessary to reduce the concentration of FCS in the medium, and put several wells in order to carry out their staining earlier than 21 days. The medium for induction is standard, I also use it.

@ Eden Klepper

Hi Eden,

Thanks for your responding, I use Polystyrene type of plates. Add 10000 cells/well and, after six days, I start to add differentiation media. They reached to near 100% confluence too.

Oksana Novikova

Thanks for your kind assistance,

I cultured the cells for 20 days. I used the regular media for six days, Then add the differentiation media for 14 days. The osteogenic differentiation started to detached after day 7.

Hi Mahsa,

Did you solve the issue with the osteogenic culture? If not, try to seed the cells on coverslips. We had the same issue and solved it using Thermanox coverslips. You struggle to get them sometimes so you can also try the Sarstedt ones: https://www.fishersci.com/shop/products/tc-coverslip-13mm-st-cs200/NC9964441

I hope it helps.