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Priming Cells with CAF or RMF CM
Priming Cells with CAF or RMF CM ( Click to Download )
ALL EXPERIMENTS INVOLVING PRIMING OF MCF7 CELLS WITH CAF OR RMF CM
- 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:
- 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).
- 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.
- After 24 hrs, wash cells twice with PBS.
- Change appropriate wells to supplemented CM.
- Add back 5% CD-FBS to thawed CM aliquots.
- Add back 2 mM L-glutamine to thawed CM aliquots.
- Spike in any other treatments, such as an anti-IL6 blocking antibody or vehicle control.
- 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.
- Take pictures after 24 hrs. Cells should look EMT’d in most conditions.
- Supplement with fresh media on day 3.
- 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).