Hi, recently I had been trying to reprogram somatic fibroblast cells from an ungulate species using chemicals following the method by Hou et al. (2013) and Long et al. (2015). Early on I did manage to get some noticeable colonies forming, however there was an incident where I added another chemical on day 16 (DZNep) that caused most of these colonies to detached, which I may assumed due to cytotoxicity (as I made a mistake in calculating the total DMSO content which exceeded 0.5%). However I tried to follow on with culturing the remaining colonies and cells, but to no avail, more and more colonies became detached, while the remaining didn't grow in size.
Before stopping the process entirely, I did live alkaline phosphatase staining to screen if these colonies were in fact iPSCs. And to my surprise, the colonies did expressed AP although faintly. I've tried to passaged these colonies but sadly they were not able to proliferate at all.
I wonder if there are any tips or methods that I could consider for these issues. Any suggestions are accepted.
Will also attach the pictures of the reprogramming process down below for your references.
1 - potential colony on day 12 (100x magnification)
2 - fluorescence image of colony on day 54
3 - brightfield image of colony on day 54
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