What is the average time, in days, that pre-adipocytes (ie - human adipose derived stromal vascular cells or ADSVC's) should be exposed to the differentiation cocktail (ie- dexamethasone, insulin, IBMX, BRL, etc.)?
Tow weeks is the customary amount of time. However, you can see changes at the gene expression level wit in a few days.
The differentiation takes about 4 days and small fat vacuoles is visible day 5 to 6. After that, it takes about two weeks to get large fat cells. We define that a cell is fully differentiated when the first vacuole is visible.
The original protocol requires dexamethasone, insulin, IBMX and indomethacin for 2 weeks and 1 week with only insulin. If you used a TZD (ie RGZ at a dose of 1 uM), cells differentiate into about 10 days.
10 days will be enough time to see fully osteogenic differentiation as assessed by Alizarin red staining
Each factor you are using has a particular window of activity with respect to the cells, that combined will result in the desired effect. You just need to know what the windows are for each factor. Suggest testing them individually, with a genetic marker to see if you are getting the desireed effect, then move on to the next factor. When you have all your information, you will have your roadmap.