I'm trying to do osteogenic differentiation of human dental pulp stem cells.
Osteogenic differentiation culture media was constituted of DMEM, 10% FBS, 50 µM ascorbate acid, 10 mM β-glycerol phosphate and 10 nM dexamethasone.
The First photo shows the control group of hDPSCs after Alizarin red S staining and second photo shows the status of DPSCs at 19 days after osteogenic differentiation and after Alizarin red S staining.
I measured alkaline phosphatase activity of groups.
The Control group was 30 U/L and OS group was >1 U/L.
your osteogenesis looks ok but Alzarin Red S staining looks problematic.
Enclosed is a reprint, pl see page 10 of my reprint
The alkaline phosphatase activity is not always in a high level, especially you culture your cells for 19 days. maybe you can measure the alkaline phosphatase activity for deferent time period.
Has your FBS been heat inactivated? We tested over 300 lots of FBS to find one lot that was absent of inductive factors. We noted that heat inactivation could be used with any FBS and negate activities of its inherent inductive factors. We used a modified heat inactivation, e.g., 8-hour incubation at 56 degree centigrade. Conversely, one can order heat-inactivated FBS from Atlas Biologicals, Fort Collins, CO.
Have you tried von Kossa staining of osteogenic aggregations with pre-treatment of controls with EGTA? von Kossa stains calcium phosphate a brown/black color (more distinctive than Alizarin Red) and EGTA will remove calcium for null staining to confirm presence of calcium.
Also appears that you have potentially adipocytic lipid vesicles in your osteogenic-induced cultures. Suggest staining with Sudan Black-B or Oil-Red O to confirm or deny their presence. Do not use any hydrocarbons (alcohol, benzene, or toluene) in processing cultures for staining, will leach out lipids prior to staining and you will get a null response.
Suggest you plate in 96-well format and retest osteogenic-inducting formulation, using concentration gradients of each ingredient. For example, dexamethasone at 10^-7 to 10^-14 concentrations. And others as well. Make the center of each concentration gradient the listed concentration of the ingredient, step 3 on one side and step 4 on the other. using a 96-well format will allow 12 experiments of 8-wells each on a single plate.
Ehsaneh Azaryan, Generally, ALP is an early osteoblast differentiation marker. We generally measure ALp activity on the 6th or 10th day of osteoblasts differentiation. we observed that at the late stage, ALP activity is significantly decreased in differentiated osteoblasts.
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