Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy
Summary: We present a protocol to induce localized DNA double-strand breaks (DSBs) in mammalian monolayer cells, using the PALM MicroBeam microdissector, followed by high-resolution visualization through expansion microscopy (ExM). We describe steps for laser microirradiation, immunostaining, and ge...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-09-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003442 |
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| author | Fernando Luna-Maldonado Diego Oliva-Rico Alejandro López-Saavedra Aquetzalli Arenas-Hernández Clementina Castro-Hernández Juan Eduardo Rodriguez Gatica Eloy-Andres Pérez-Yepez José Díaz-Chávez Luis A. Herrera |
| author_facet | Fernando Luna-Maldonado Diego Oliva-Rico Alejandro López-Saavedra Aquetzalli Arenas-Hernández Clementina Castro-Hernández Juan Eduardo Rodriguez Gatica Eloy-Andres Pérez-Yepez José Díaz-Chávez Luis A. Herrera |
| author_sort | Fernando Luna-Maldonado |
| collection | DOAJ |
| description | Summary: We present a protocol to induce localized DNA double-strand breaks (DSBs) in mammalian monolayer cells, using the PALM MicroBeam microdissector, followed by high-resolution visualization through expansion microscopy (ExM). We describe steps for laser microirradiation, immunostaining, and gel embedding. The protocol enables 4.5-fold physical expansion and confocal imaging of DNA damage sites. We quantify nuclear and damage site expansion using Fiji by measuring nuclear area and cross-sectional fluorescence profiles. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-bcceea30325d4ee6a7643bb175f7ba1c |
| institution | Matheson Library |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-bcceea30325d4ee6a7643bb175f7ba1c2025-07-11T04:31:45ZengElsevierSTAR Protocols2666-16672025-09-0163103938Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopyFernando Luna-Maldonado0Diego Oliva-Rico1Alejandro López-Saavedra2Aquetzalli Arenas-Hernández3Clementina Castro-Hernández4Juan Eduardo Rodriguez Gatica5Eloy-Andres Pérez-Yepez6José Díaz-Chávez7Luis A. Herrera8Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, Mexico; Instituto Nacional de Cancerología, Advanced Microscopy Applications Unit (ADMiRA), Av. San Fernando 22, Col. Sección XVI, Tlalpan 14080, CDMX, MéxicoUnidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, MexicoUnidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, Mexico; Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Calle del Puente #222, Col. San Bartolo el Chico, Tlalpan 14380, Ciudad de México, México; Instituto Nacional de Cancerología, Advanced Microscopy Applications Unit (ADMiRA), Av. San Fernando 22, Col. Sección XVI, Tlalpan 14080, CDMX, MéxicoInstituto Nacional de Cancerología, Advanced Microscopy Applications Unit (ADMiRA), Av. San Fernando 22, Col. Sección XVI, Tlalpan 14080, CDMX, MéxicoUnidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, Mexico; Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City 04510, MexicoInstitute for Experimental Epileptology and Congnition Research, University Hospital Bonn, Venusberg-Campus 1, 53127 Bonn, GermanyLaboratorio de Genómica, Instituto Nacional de Cancerología, Av. San Fernando 22, Col. Sección XVI, Tlalpan 14080, CDMX, MexicoUnidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, Mexico; Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Calle del Puente #222, Col. San Bartolo el Chico, Tlalpan 14380, Ciudad de México, México; Corresponding authorUnidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan)- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City 14080, Mexico; Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Calle del Puente #222, Col. San Bartolo el Chico, Tlalpan 14380, Ciudad de México, México; Corresponding authorSummary: We present a protocol to induce localized DNA double-strand breaks (DSBs) in mammalian monolayer cells, using the PALM MicroBeam microdissector, followed by high-resolution visualization through expansion microscopy (ExM). We describe steps for laser microirradiation, immunostaining, and gel embedding. The protocol enables 4.5-fold physical expansion and confocal imaging of DNA damage sites. We quantify nuclear and damage site expansion using Fiji by measuring nuclear area and cross-sectional fluorescence profiles. : 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/S2666166725003442Cell BiologyCancerMicroscopy |
| spellingShingle | Fernando Luna-Maldonado Diego Oliva-Rico Alejandro López-Saavedra Aquetzalli Arenas-Hernández Clementina Castro-Hernández Juan Eduardo Rodriguez Gatica Eloy-Andres Pérez-Yepez José Díaz-Chávez Luis A. Herrera Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy STAR Protocols Cell Biology Cancer Microscopy |
| title | Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy |
| title_full | Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy |
| title_fullStr | Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy |
| title_full_unstemmed | Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy |
| title_short | Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy |
| title_sort | protocol for induction and study of dna double strand breaks in mammalian cells using palm microdissection and expansion microscopy |
| topic | Cell Biology Cancer Microscopy |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725003442 |
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