I have tried to establish colonies from mouse iPSCs. However, after I pick up good colonies to establish in 24 well plate I can not be succesful and they die or undifferentiate. I have attached some colony pictures from my passage 1 or 2 iPSCs to make sure apperance of these colonies.
I'm not sure of your question in relation to those photos. If these are photos of passaged colonies, then that would suggest they are surviving passaging.
Your feeders are more dense than they need to be and both of those colonies are quite small. Is that the size that they are when you passage them? How are you passaging them.
Thank you for answering me. I would like some clarification about my cells apperance . These are passage 2 ipsc colonies derived from KO mouse tail fibroblasts. To establish ipsc clone i pick up like these colonies. After pick up i put these colonies on one well/24 well plate' but they do not survive. My question is that these are correct colonies for picking up or have I different mistakes during this process??
They are distinct colonies to pick up, but if these are colonies that have arisen from reprogramming and are about to be picked as independent clones for the first time, then I would say that they still are too small to pick yet. Have you tried waiting until the colonies are larger?
I suppose we simply are using a different terminology, but if these have not yet been picked from the original reprogramming, then I would not say that they have any passage number yet.
Thank you so much. Maybe my terminology would be wrong. After reprogramming to establish clone I choose individual colony (like in picture) to pick up. During these process I keep always my heterogenous colonies. Because after pick up like these colonies to establish clone, they die or differentiate. I checked many papers, mouse iPSCs so different from human iPSCs regarding size and apperance. The another reason why I choose like these small colonies , they quickly differentiate maybe in 2 or 3 days.
OK, so these are colonies before picking. I was confused by the P1 or P2 terminology, since I don't assign a passage number to an iPSC until the first split AFTER picking. Certainly there are different conventions for this and your terminology is as good as mine. I just wanted to make sure what I was looking at.
I've done very little with mouse iPSCs and it was years ago. I let them get bigger than those before picking but they certainly were smaller than the usual size that I pick human iPSCs.
I am concerned if yours are differentiating so quickly, forcing you to pick them (relative to my experience) so early. What are you using for a reprogramming media? Does it differ from your stem cell maintenance media?