A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy.

ABSTRACT: Microscopic cell segmentation typically requires complex imaging, staining, and computational steps to achieve acceptable consistency. Here, we describe a protocol for the high-fidelity segmentation of the nucleus and cytoplasm in cell culture and apply it to monitor interferon-induced signal transducer and activator of transcription (STAT) signaling. We provide guidelines for sample preparation, image acquisition, and segmentation. The approach performs indistinguishably from neural-network-based segmentation while requiring only conventional and cost-effective techniques. The protocol can be adapted to other signaling molecules undergoing nucleo-cytoplasmic shuttling and to high-throughput applications. This protocol enables simultaneous monitoring of two STAT isoforms using only convent

SUBMITTER: Szanda G 

PROVIDER: S-EPMC11969409 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature