Protocol for analyzing autophagy-related cellular phenotypes in autophagy-deficient human embryonic stem cell-derived lacrimal gland-like organoids.

Autophagy is a vital catabolic process that maintains cellular homeostasis and survival and supports gland tissue development and function. Here, we present a protocol to characterize the cellular phenotypes of autophagy-deficient (ATG5-/-) lacrimal gland (LG)-like organoids derived from human embryonic stem cells. We describe steps for organoid cryosectioning, followed by the quantitative assessment of cell death and aggresomes using fluorescence-based TUNEL and ProteoStat staining, respectively, along with pharmacological rescue. This approach enables robust evaluation of autophagy-related cellular dysfunction in human organoid models. For complete details on the use and execution of this protocol, please refer to Kocak et al.1.