Hello everyone, I am trying to access TNFalpha cytotoxic effect on A549 cell line ( from 2.5-40ng/ml) using Invitrogen Live/Dead staining protocol (Calcein AM/ Ethidium homodimer-1). I found that TNFalpha appears to significantly reduces A549 proliferation, but the results obtained from the live/dead staining is only 2-5% difference in number of dead cells. I could not find data performed similar staining on A549, so I wonder is it possible for TNFalpha to reduce proliferation without inducing cell death? I seeded the cells with TNF incubation in 24 well-plates, and the cells reached 70-90% confluency by the time of staining.