Dear all,

I am isolating mesenchymal stromal cells (MSCs) from the bone marrow of adult C57BL/6 mice.

The culture conditions are:

-IMDM + L-glut, 20% of 50:50 FBS:HS, 1% P/S

-Medium replaced each 3-4 days

-When confluent, adherent cells were trypsinized and subcultured (process for MSCs purification)

When I obtain a pure MSCs population, I seed 7000 cells par well in a 96-well plate and I induce osteogenesis in the following way:

-osteogenic medium (50 µg/ml ascorbic acid, 10 mM β-glycerophosphate) in IMDM + L-glut, 20% of 50:50 FBS:HS, 1% P/S. )

-The medium is changed every 3 days

-Alizarin Red staining is performed 21-25 days upon induction of differentiation (after PFA fixation) to evaluate mineralization

The experiment seems to work as I obtain a nice Alizarin Red staining (see attached Picture 1). However, I am curious in the morphology of the cells I obtain. As you can see both from Picture 2, 3, 4 and 5 I have plenty of these round-shape cells that stain for DAPI (Picture 4). Does anyone have experience observing this kind of cells? Are these osteoblasts? They are also weakly stained for Alizarin Red, as you can see from picture 5, or is this aspecific?

I thank you in advance for your help

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