Hi everyone. I am culturing A549 in F12K suplemented with 10% FBS and 1% PS. After staining cells mark very positive for SMA and almost no E-cadherin without the addition of TGF-B1. Why is this happening. I am using a P2 that was expanded for 4 days and then I cultured in well plates for 4 days. Should I use low serum media like (2%) at all time. I am aware that FBS may content TGFB but I am afraid cells wont survive in low serum. What are the right conditions for A549 culture? It seems they are very sensitive to EMT. There is cronctadictory information out there, people use complete media, some times starve cells for short periods. Is this starving able to reverse EMT? On the other hand is confluency inducing also EMT or low density may also induce it? Please, I am quit lost here.