Osteoblast differentiation assay

1. Plate cells in culture dish (1 x 10⁵/ 6 well plate for stromal cells).  Need two wells per condition for staining, more if you want to collect RNA.
2. Allow cells to grow to confluence.
3. Once cells have been at confluence for 2 days begin adding differentiation media-regular growth media plus:
   0.01M β-glycerol phosphate
   100mM Ascorbic Acid
4. Add differentiation media to one well and regular growth media to other well so that you have an undifferentiated control well of cells.
5. Refeed every 2-3 days, making fresh differentiation media right before adding.
6. Mineralization should occur around 17-19 days
7. Stain cells treated with control and differentiation media with Alizarin Red S on day 21.
8. Image cells
9. Collect RNA if you set up enough wells to do so.  Check changes in gene expression (these should all increase if differentiation occurred):

AP

RunX2

Osterix

Osteocalcin