Abstract: The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is\nlargely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug\nscreening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged\nto be a better model of the in vivo environment, but detailed cell death assays for 3D cultures\n(including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis\nmethod, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types\nincluding ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses\nSytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in\nspheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton\nX-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from\nall cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that\n2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death\ninduction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death\ntype in 3D cultures, including spheroids.
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