Gergő Szanda, Éva Wisniewski, László Barna, Gábor Turu, Ken Mackie
{"title":"A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy.","authors":"Gergő Szanda, Éva Wisniewski, László Barna, Gábor Turu, Ken Mackie","doi":"10.1016/j.xpro.2024.103588","DOIUrl":null,"url":null,"abstract":"<p><p>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.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103588"},"PeriodicalIF":1.3000,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2024.103588","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
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.