Feeder cultures - Now we're back online- I could do with some advice (Jan/28/2009 )

*Tears of joy and relief flowing down my face*

Its back!

Anyway,

I have some cells that I need to culture with a feeder layer- I have irradiated fibroblasts and keratinocyte cell line. Since the fibroblasts are irradiated I have to replace them every 2-3 weeks, but my keratinocytes should be confluent before then.
Do any of you think I can seed my fibroblasts in a few different flasks- to act as a stock for the keratinocyte cell line or will I have to seed fresh fibroblasts each time before spliting my keratinocytes?

Any advice- I've never used a feeder layer before and can't afford to buy these cells for a while so I don't want to mess things up.


Lost

I have work a little with feeder layers. I didn't irradiated the cells, I used mitomycin to inactivate their growth. I froze little aliquots of my fibroblast cell line and I only treated the cells when I need, after thaw them and seed.
I supose your cells irradiated didn't growth, but I don't know if you could mantain them in culture for long time.
If I were you I would buy a fibroblast cell line (I used 3T3-J2 from mouse) and then you work with them as usual and only irradiate o treat with mitomycin when necesary . After that, you add your keratinocytes over your feeder layer until confluence.
Best,
Gugo

gugo on Jan 30 2009, 11:10 PM said: