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

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 conventional techniques and equipment and improves upon the assay published in Szanda et al.1. Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Citation

Gergő Szanda, Éva Wisniewski, László Barna, Gábor Turu, Ken Mackie. A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy. STAR protocols. 2025 Mar 21;6(1):103588

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PMID: 39862428

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