Primary Dermal Fibroblast isolation - (Jul/16/2012 )

Hi guys,

Lately when I isolate mice DF, the yield of cells is low and cells won't proliferate. The fibroblasts become large and striated (senescence).

I use this protocol: http://www.mechamlab.wustl.edu/Lab%20Web%20Page.data/Library/MethodsPDFs/Mouse_Skin_Cells.pdf

However at the end I do an extra spin step to remove the collagenase.

Do you guys have any solution or ideas?

Thanks!

However right now I am not sure whether these cells are going senescent since these cells are only Passage 0-1. So I am thinking that these cells might be differentiating to myofibroblasts...

Anyone has suggestions on this or how to prevent this?