I found some protocols on the internet and I tried them out, but they don't seem to be working. I separated the cortex from the kidney and minced and then digested the tissue with HBSS buffer containing the collagenase for 20 minutes 3 times. However, after 24 hours, I didn't see many cells attaching.
Do I need ot use a collagen-coated plate or a plate covered by another material?
If I use percoll to separate the living cells from the dead ones, what percentage of percoll should I use?
Hi Xiaoyan,
I work with pig lens and ciliary epithelial cells and I frequently establish primary culture. To establish a good healthy epithelial culture selecting proper epithelial growth media and supplements is a must. Some times epithelial growth supplements help a lot to have a good culture.
Sciencell has a varieties of epithelial growth media and supplements to select on for your purpose. If you want to give a try without buying any commercial formulation you can try the following.
1. Use Poly-L-Lysine coated dishes/plates
2. Try varying serum concentration
3. Increase the seeding density (15X10^3-25X10^3/cm2)
4.Perform centrifugation and the cell suspension preparations at 2-4 deg C
5. Add EGF (5ng/ml) in the culture media
6. Do not touch or move the dish/plate for 24h after seeding
For a generalized protocol for establishing Renal Inner Medullary Collecting Duct Epithelial Cells you might consider reading the article I am sharing.
Hope that helps!
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