Protocol to quantify LRRK2-mediated Rab10 phosphorylation using high-content imaging

Rab10 phosphorylation at Thr73 (pRab10) is a well-established readout of the kinase LRRK2, a protein whose mutations are associated with Parkinson's disease. Here, we present a protocol for high-content quantification of endogenous pRab10-positive vesicles in primary astrocyte-enriched cultures using the Operetta CLS system. We describe steps for cell culture, immunofluorescence, image acquisition, and quantitative analysis using Harmony software. This protocol is applicable to other LRRK2-expressing cells and offers a scalable platform for quantitative studies of LRRK2 physiological and pathological activity.