mouse embryonic stem cell culture on gelatine coated plates - (Apr/13/2014 )

Hi everyone,

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!!

Thank you!

Elisa

You'd better seed some MEFs in the dish after coating gelatin. it will make mESs grow in clumps. You said that  in 2days cells should reach about 70% confluency. it is decided by the density of mESCs you subcultured. if you seed more cells, i will take less time to reach  70% confluency. I think 4 days is okay as long as the morphology is normal.