Measuring cell survival and cell death

Cell survival was measured with CellTiter-Glo (Promega, G7570) luminescent viability assay according to the manufacturer’s instructions. Membrane leakage of necroptotic cells was measured by CytoTox-Glo (Promega, G9292) luminescent assay according to the manufacturer’s instruction. Briefly, cells were plated in 96-well culture plate at a concentration of 5000 per well 24 hours before the assay. After inducing necrosis, the plates were moved out of the incubator and allowed to reach room temperature. A total of 50 μl of either CellTiter-Glo or CytoTox-Glo reaction solution was added into each well and mixed with cell culture medium by pipetting. The reaction was carried out on a horizontal shaker at room temperature for 15 min. Luminescent signal was then measured in a plate reader (PerkinElmer, EnSpire). Necrotic cell death was also measured in live cells by applying propidium iodide in culture medium and capturing by spinning disk confocal microscope.