{"doi":"10.1016/j.xpro.2024.103588","title":"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 conventional techniques and equipment and improves upon the assay published in Szanda et al. 1 • Steps to reliably stain nucleus and cytoplasm in cell cultures • Guidance on high-fidelity segmentation of nuclear and cytoplasmic regions • Instructions on monitoring the activity of two STAT isoforms simultaneously • Adaptable to various signaling pathways with only standard reagents and equipment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.","journal":"STAR Protocols","year":2025,"id":560839,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9513,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1118873,"name":"Éva Wisniewski","orcid":"0000-0001-8698-6867","position":1,"is_corresponding":false},{"id":663240,"name":"L Barna","orcid":"0000-0002-3856-5774","position":2,"is_corresponding":false},{"id":12514,"name":"Gábor Turu","orcid":"0000-0002-4421-3812","position":3,"is_corresponding":false},{"id":227564,"name":"Ken Mackie","orcid":"0000-0001-8501-6199","position":4,"is_corresponding":false},{"id":1118872,"name":"Gergő Szanda","orcid":"0000-0002-1308-7593","position":0,"is_corresponding":true}],"reference_count":26,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:55:46.921885Z","pmid":"39862428","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}