I want to check role of Wnt signaling during differentiation of cardiosphere derived cells to cardiomyocytes. Wnt acts in time-dependent manner during differentiation. So, I want to add Wnt inhibitor at different time points of differentiation induction. Is IC50 value to be considered ? or have to check the expression of markers at different time periods of differentiation with different concentrations of inhibitor.
Kindly provide with proper protocol to determine the concentration
Thank you
I think it is a better approach if you set a time when you observe clear changes on differentiation markers. Moreover, you must calculate the inhibitor IC50 and then use 2, 4 and 8X this concentration at a short time point already decided. Now you will be able to analyse if the changes in the differentiation markers are inhibitor dose-dependent.
The basic biological concepts in life process including cellular differentiation is very simple on molecular accepts but not in signaling levels. Deriving the value in units and determining the concentration of inhibitor is surely vary from the age of your cells and obviously the quality. My understandings if not wrong, please start with more than 12X and check it. Importantly keep remember the medium disturbances too, because there are many bond breaking events and joining too.
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