Fluorescence Activated Cell Sorting (FACS)

For control melanoma cells, tumor cells were seeded onto rigid plastic at ~ 20% density to avoid overcrowding of cells. 12 hrs later, 0.1% FBS containing medium was switched to culture the tumor cells up to 24 hrs to synchronize the cell cycle; for TRCs, cells grew in soft fibrin gels for 4 days, then 0.1% FBS containing medium was changed to culture the TRCs up to 24 hrs to synchronize the cell cycle. TRCs or control melanoma cells that were serum deprived for 0 hr, 12 hrs or 24 hrs were collected, and the cell cycle of all samples was analyzed using flow cytometry.