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mouse embryonic stem cell culture on gelatine coated plates

stem cell gelatin

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#1 Elisa90

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Posted 13 April 2014 - 03:38 PM

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



#2 Jane1207

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Posted 30 April 2014 - 10:10 PM

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. 






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