Protocol for quantifying aberrant features of adult Drosophila midgut stem cells as tumor-prediction biomarkers

Here, we present a protocol for quantifying aberrant features of clustered intestinal stem cells (ISCs) in the adult Drosophila midgut, enabling tumor-prediction biomarker assessment. We describe steps for clustered ISC labeling, immunostaining, and cell number and size enumeration per midgut region during aging. We then detail procedures for determining mitosis rate and ploidy per clustered ISC. These measurements can be compared across different genotypes and experimental conditions and correlated with the corresponding late-in-life tumor incidence. For complete details on the use of this protocol, refer to Neophytou et al.¹.