Collagen coating of glass bottom dishes and glass bottom plates
Materials
Rat tail collagen (type I) solution
Rat tail collagen I is available from many commercial vendors, such as BD Biosciences(cat# 354249), Sigma (cat #C3867), Invitrogen (cat #A10483-01).
It is recommended to purchase collagen solutions, so that sterilization is not needed since filter sterilization of collagen could be problematic.
Dilute the stock solution to 50 ug/ml with 0.02M HAc or 0.01M HCl.
Procedure
- Add the collagen solution (50 ug/ml) into a glass bottom dish or glass bottom plate at 5 ug/cm2. If cells will be cultured only on the glass surface, then only the glass surface needs to be covered with collagen solution.
- Incubate for 1 hour at room temperature in a hood.
- Carefully aspirate the collagen solution.
(alternatively, the glass bottom dish/plate can be left in the hood to dry overnight, and the following washing step is not needed) - Rinse 3 times with PBS or serum-free media.
- Collagen coated glass bottom dish or glass bottom plate can be kept at 4℃ for about a month. Note that coating on glass surface is less stable than coating on plastic tissue culture dishes or plates. Thus it is recommended to coat the glass bottom dish/plate right before cell culture.
Alternative Procedure
Some people recommend coating with poly-l-lysine first before coating with collagen, view this video for more details.
