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Year:: 2022
Contributor:: Yongkuan Zhang (creator); Nicola Neretti (advisor); Miiko Sokka (advisor); Brown University. Undergraduate Research Assistantship in Molecular Biology, Cell Biology, and Biochemistry (MCB) (research program)
Genre:: posters
Subject:: Biology; Aging; Molecular biology; Biochemistry
Extent:: 1 poster
DOI: https://doi.org/10.26300/xda7-9h81

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Abstract:: Cellular senescence is a state of permanent cell cycle arrest that plays a major role in damage response, tumor suppression, and aging. Although many prominent phenotypes of senescent cells have already been identified, compiling a comprehensive list of biomarkers that characterize and define the senescent state is still an ongoing challenge. By analyzing chromosomal architecture, damage signaling, and nuclear structure, the Neretti lab seeks to explore how alterations of these biomarkers in senescence cells can aid in their distinction from regular cells. By combining two techniques: Iterative Indirect Immunofluorescence Imaging (4i) and Fluorescence in-situ hybridization (FISH), antibody staining and hybridization of DNA probes were achieved in the same cells. To characterize biomarkers in senescent cells, we compared etoposide-treated and untreated human fibroblast (BJ-hTERT) cells, with etoposide acting as a DNA damaging agent used to induce senescence. Our selection of biomarkers includes DNA damage, nucleolar morphology, and genomic markers. The innovative imaging buffer used in 4i allowed for gentle elution of antibodies without damaging the overall cellular structure, therefore making multi-round antibody staining as well as combination with FISH possible. Repeated rounds of imaging using fluorescent microscopy were performed and the samples were eluded and re-stained after each round. The probes selected for FISH targeted the alpha satellite as well as the telomeric regions to investigate whether changes in repetitive segments of DNA can be used as markers to identify senescent cells. This novel merging of existing techniques allows for the targeting of genetic and antigenic biomarkers within the same cells. Analysis on the subcellular, single-cell, and population-level using this technique can not only improve the identification of senescence cells but also further our understanding of their unique role in aging and disease.

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Yongkuan Zhang, "Multi-round Immunofluorescence imaging combined with Fluorescence in-situ hybridization allows multiplexed characterization of senescent cells" (2022). Summer Research Symposium. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/xda7-9h81

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Summer Research Symposium

Each year, Brown University showcases the research of its undergraduates at the Summer Research Symposium. More than half of the student-researchers are UTRA recipients, while others receive funding from a variety of Brown-administered and national programs and fellowships and go …
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Multi-round Immunofluorescence imaging combined with Fluorescence in-situ hybridization allows multiplexed characterization of senescent cells