I'm generating mammospheres from MCF7 lines in phenol-red free DMEM/F12, 20 ng/mL of EGF, 10 ng/mL of bFGF and 1X ITS-A (Gibco; 51300-044) on Nunclon Sphera 24-Well Plate. And before, I'd see mammosphere formation within 7 days.
Recently, I'm using an old stock of phenol-red free DMEM/F12, I changed stock bFGF and changed to 1X ITS-G (Gibco, 41400-45) and I don't see mammosphere formation.
What should I change or how do I go about troubleshooting?