3 replies to this topic

[sabi67]

Hi,

I plan to prepare conditioned media from tumor cells for later use in culture on stroma cells to perform different assays. I was told to add protease inhibitor to the conditioned media before freezing it at -80 C. That surprises me because I would add protease inhibitor if I wanted to extract proteins but not when using it again in culture, I think proteases inhibitor could interfere with the effect the conditioned media will have or not when I will use it in culture. Anybody has an idea if I shoud effectively add protease inhibitor or not?
thanks a lot for your help

Edited by sabi67, 07 November 2009 - 03:51 PM.

[klinmed]

sabi67, on Nov 8 2009, 12:16 AM, said:

Hi,

I plan to prepare conditioned media from tumor cells for later use in culture on stroma cells to perform different assays. I was told to add protease inhibitor to the conditioned media before freezing it at -80 C. That surprises me because I would add protease inhibitor if I wanted to extract proteins but not when using it again in culture, I think proteases inhibitor could interfere with the effect the conditioned media will have or not when I will use it in culture. Anybody has an idea if I shoud effectively add protease inhibitor or not?
thanks a lot for your help

If your CM is made in medium containing serum (even as low as 0.1 %), there should be no need to add inhibitors. Serum contains many protease inhibitors (alpha1-antitrypsin, alpa2-macroglobulin etc).

[skeuos]

sabi67, on Nov 8 2009, 12:16 AM, said:

Hi,

I plan to prepare conditioned media from tumor cells for later use in culture on stroma cells to perform different assays. I was told to add protease inhibitor to the conditioned media before freezing it at -80 C. That surprises me because I would add protease inhibitor if I wanted to extract proteins but not when using it again in culture, I think proteases inhibitor could interfere with the effect the conditioned media will have or not when I will use it in culture. Anybody has an idea if I shoud effectively add protease inhibitor or not?
thanks a lot for your help

Klinmed's right about serum having some inhibitors already, but the more important issue is as you suggest - proteases are important for activating and freeing growth factors and other molecules. Adding inhibitors would interfere with this.

[sabi67]

Thank you very much for your answers! It helped me a lot