I have ordered MCF10A cells from AddexBio 2 years ago and grew them in 1:1 DMEM/F12, 5% Horse Serum (Wisent), 10ug/mL Insulin, 0.5ug/mL Hydrocortisone, and 20ng/mL EGF. They looked great, froze and thawed great, and I induced EMT using recombinant hTGFB-1 (Verified by markers using RT-qPCR, WB, then RNAseq and ATACseq).
When I ran out of Horse Serum, I ordered a new batch and all my cells suddenly died. I called the company and they did not have another batch to provide to me for testing, they suggested using FBS instead. However with FBS, the cells look more mesenchymal and don't respond to TGFb treatment (no change in ECAD, NCAD, VIM, SNAIL signal in RT-qPCR or WB). I also tested the heat-inactivated Horse Serum from Wisent and had the same issue. I switched companies and tested HI and non-HI Horse Serum from Gibco, but the cells still look fibroblastic and have no change in marker expression.
I have re-ordered and made fresh stocks of all media additives, and have ordered TGFb from 2 different companies. Every time I try a new media, I start from a fresh vial of early passage cells I froze when I had epithelial-looking cells. Has anyone ever experienced this, or would anyone have any idea of things I could do?
Thanks in advance
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