Collecting CM From CAFs or RMFs ( Click to Download )

1. Seed fibroblasts at one of the following cell densities depending on cell number: 100% confluence, 10⁶ cells per 10 (dense) or 15 cm (sparse) plate, or 5×10⁶ cells per 15 cm plate. Be consistent if comparing CM potencies among various patient fibroblasts. Try to match passage number if possible, although this is not always possible.
   1. I use PRF-DMEM + 2% CD-FBS + 2 mM L-glutamine + AB/AM for a 3 day CM collection (i.e. collect CM after 72 hrs).
   2. For CM analysis by WB or ELISA, it is better to use S/S CM. For this, use PRF-DMEM + 0.5% CD-FBS + 2 mM L-glutamine + AB/AM for a 24-48 hr CM collection (72 hrs and the cells start to look pretty miserable).
2. If administering a treatment (such as PGE2 or EtOH vehicle control), add this at the time of seeding in PRF-DMEM conditions. I use PGE2 at 0.5 µM. I am able to still see a phenotype by only adding PGE2 1X (at start of experiment) over 72 hrs, but some say PGE2 is unstable; may want to supplement with fresh PGE2 every day and see if effect is more robust, if this is a concern.
3. At 72 hrs, harvest CM from cells and filter using a Steriflip 0.22 µm filter. Aliquot CM at volumes that will be used for a particular assay. If CM is being used to prime MCF7 cells, typically 12 mL aliquots will suffice. Do not F/T and try to reuse for another assay.
4. Store all CM at -80°C.