I have been treating my SH-SY5Y cells with MG132 for 24 hr to induce cell death. A while ago my EC50 that kills 50% of the cells was determined around 2.5uM but for some reason now I find it around 500uM at the same cells, techniques and detection kit but I don't have a clue why. Cell death is assessed by LDH assay (by Promega's CytoTox-ONE™ Homogeneous Membrane Integrity assay). I have always been using new MG132 product. The morphology of cells are quite dead-like already from 10uM but I know that membrane integrity may be maintained until the last minute. Could someone possibly help with this or share what your EC50 is in this cell line? Thanks.