Priming Cells with CAF or RMF CM ( Click to Download )

• Primed cells can be further analyzed by qPCR/WB, sphere formation, and in vivo tumor formation
• To control for differences in CAF and RMF passage number, and any differences that might ensure because of this, it is best to seed a large number of cells for priming at the beginning and then disperse among assays:

To Prime Cells:

1. Seed appropriate number (if using the experimental setup described above, seed at least 10⁶) of MCF7 (preferably low passage) cells to either 10 cm plates (for 10⁶ cells) or 6 well plates (for lower dilutions) in their normal growth media (DMEM + 10% CS + AB/AM).
2. The next day, thaw CM from -80 freezer along with an aliquot of charcoal/dextran stripped FBS (CD-FBS) and 200 mM L-glutamine. Thaw only what you need- do not freeze/thaw CM.
3. After 24 hrs, wash cells twice with PBS.
4. Change appropriate wells to supplemented CM.
   1. Add back 5% CD-FBS to thawed CM aliquots.
   2. Add back 2 mM L-glutamine to thawed CM aliquots.
   3. Spike in any other treatments, such as an anti-IL6 blocking antibody or vehicle control.
   4. For wells receiving regular media (not CM), use PRF-DMEM + 5% CD-FBS + 2 mM L-glutamine + AB/AM with any additional treatments, such as 0.5 uM PGE2 or vehicle control.
5. Take pictures after 24 hrs. Cells should look EMT’d in most conditions.
6. Supplement with fresh media on day 3.
7. Prepare for flow cytometry on day 6 (see protocol Flow Cytometry for CD44+/CD24-/EpCAM+ cells), RNA isolation (use Qiagen kit), or western blot (for RIPA extraction).