conditioned medium - (May/21/2019 )

Hi, does anyone know ho wto prepare conditioned medium (CM) and add to other cells.

A. This is what I understand based on what I've read:

1. Grow cells in 10cm dish 

2. after 3 days when cells have reached 70-80% confluence, collect medium

3. centrifuge collected medium at 1,000 g for 10mins to remove cell depris etc  and store in 1ml aliquots in -80deg?

Am'I misisng any step.

B. When we use the CM, do we have to dilute with regular medium in 30%, 50% ratio or use CM directly?

Thank you

You pretty much have it correct. I would recommend filtering the medium through a 0.1 um filter too, just in case there is some contamination associated with the feeder cells.

You can dilute, but it is not absolutely necessary in my experience, and will depend on what exactly you are doing with the cells.