A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy
Summary: 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 sign...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724007536 |
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| author | Gergő Szanda Éva Wisniewski László Barna Gábor Turu Ken Mackie |
| author_facet | Gergő Szanda Éva Wisniewski László Barna Gábor Turu Ken Mackie |
| author_sort | Gergő Szanda |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-f0ad438c6e3e48d3bb4def603906957b |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-f0ad438c6e3e48d3bb4def603906957b2025-01-26T05:04:56ZengElsevierSTAR Protocols2666-16672025-03-0161103588A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopyGergő Szanda0Éva Wisniewski1László Barna2Gábor Turu3Ken Mackie4Gill Institute for Neuroscience, Program in Neuroscience, Department of Psychological and Brain Sciences Indiana University, Bloomington, IN 47405, USA; Department of Physiology, Semmelweis University Medical School, Budapest 1094, Hungary; Corresponding authorGill Institute for Neuroscience, Program in Neuroscience, Department of Psychological and Brain Sciences Indiana University, Bloomington, IN 47405, USA; Department of Physiology, Semmelweis University Medical School, Budapest 1094, HungaryAddiction and Neuroplasticity Lab, Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405, USADepartment of Physiology, Semmelweis University Medical School, Budapest 1094, Hungary; Institute of Molecular Life Sciences, Centre of Excellence of the Hungarian Academy of Sciences, HUN-REN Research Centre for Natural Sciences, Magyar Tudósok krt. 2., Budapest 1117, HungaryGill Institute for Neuroscience, Program in Neuroscience, Department of Psychological and Brain Sciences Indiana University, Bloomington, IN 47405, USA; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724007536Microscopysignal transductionmolecular/chemical probes |
| spellingShingle | 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 Microscopy signal transduction molecular/chemical probes |
| title | A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy |
| title_full | A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy |
| title_fullStr | A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy |
| title_full_unstemmed | A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy |
| title_short | A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy |
| title_sort | 2d cell segmentation protocol for monitoring multiple stat signaling pathways by fluorescence microscopy |
| topic | Microscopy signal transduction molecular/chemical probes |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724007536 |
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