Protocol for phenotypic screening of human intestinal organoids using TNF-induced intracellular ATP changes as a viability readout

Phenotypic screening is a powerful strategy for identifying bioactive compounds in physiologically relevant systems. Here, we present a protocol for evaluating tumor necrosis factor (TNF)-induced changes in intracellular ATP as a viability readout. We describe steps for organoid expansion in type I collagen gel, recovery of organoids, dissociation into a homogeneous single-cell/small-cluster suspension, TNF stimulation and compound treatment, and measurement of intracellular ATP levels. This protocol addresses variability arising from the three-dimensional nature of organoids and introduces strategies to minimize such variability. For complete details on the use and execution of this protocol, please refer to Takahashi et al.¹.