Dear all,
I need to differentiate osteoblasts and osteoclasts from mouse bone marrow and I am following this detailed protocol: Article Isolation, Culture, and Differentiation of Bone Marrow Strom...
On step 7, for osteoclasts differentiation I don't know what to expect from the cells.
On J0, I plated all the cells in a flask.
After 48h, I collected the adherent cells and I seeded them in 6-well plate for osteoblast differentiation.
I also collected the non-adherent cells and I seeded them in 6-well plate for osteoclast differentiation. In the protocol, They imply that the non-adherent cells will adhere and I should use this now adherent cells for osteoclast differentiation. After how many day will this "non-adherent' cells will become adherent cells? Are you all using a similar protocol?
Thank you very much for you help.
Gabrielle
Hi Gabrielle! I usually culture my non-adherent cells for 12 days with media changes every 2-3 days. The first time I fixed them, I thought they would all wash away but these cells tend to be quite robust in my experience and adhere nicely!
Thank you very much for your help!
Do you directly culture the non-adherent cells with osteoclast differentiation media since Day 1? Or do you differentiate the cells after the 12 days?
I give the differentiation media at Day 1, right after I collect the non-adherent cells! At around 9 days, I will look for mature osteoclasts and take it out to 12 days if necessary.