I am new to stem cell culture; right now I am working with mESCs (R1 line) and I am culturing them on gelatine-coated plates, using serum and LIF.
I have a couple of general questions:
1. The morphology of the cell puzzles me: some grow in clumps, while others are flat and spread-out.
2. They grow much slower than they are supposed to: the protocol says that in 2days they should reach about 70% confluency, but mine need about 4 days.
3. I am using 0.2% gelatin, but I keep getting floating cell aggregates. What does this mean? Is my coating time too short? Is the gelatine concentration to little and should I increase it?
4. I was researching online the shelf-life of gelatine in solution stored at 2-8*C and I found a couple of suppliers sheets that state that it is indefinite. is this true?
I would appreciate any thoughts/comments/suggestions you guys have, I could really use some help!!