Mouse Renal fibroblast Primary Culture - (Jul/22/2009 )

Hi everyone this is my first post ,

I have a few questions about my research and this looks like a really good site so here it goes.
For my Masters I have to culture renal fibroblasts from mice and we have been able to culture the cells to passage 3 but no further- a T75 at passage 1 can give 5 million but we would only get a few hundred thousand cells at passage 2 even though both may look confluent-does anyone else have any experience culturing these cells? any tips would be really appreciated

Also, I have done a lot of FACS on these cells but can't find a marker to identify the fibroblasts so that I know we have fibroblasts not epithelial cells or any other type-would anyone be aware of marker(s) that might be useful to stain for?

Last question- I am going to do some FACS and immunocytochemistry for alpha smooth muscle actin (ACTA) so I can identify myofibroblasts and I will need a postive control- I know most muscle cell types stain for ACTA so I was going to go with skeletal muscle, but I have no idea how to prepare/grow a primary culture of these cells from a mouse.
I'd really be grateful for any help or advice

thanks

Primary culture is difficult and time consuming - primary cells will not grow in an unlimited fashion like most cell lines and will senesce after a few cell divisions. They will also be contact inhibited in most cases, so if you let your culture become confluent, the cells will stop growing and may not start growing again when passaged. You may need to play around with higher FBS concentrations, supplementing with growth factors, CO₂%, type of medium etc. Have a look in a good cell culture manual such as "Culture of Animal Cells" by R.I. Freshney for more hints and instructions.

ok all good tips!-thanks for your help I'l give it a shot