Cellular senescence is a mechanism characterized by the irreversible arrest of cell division caused by DNA damage. Accumulation of senescent cells in the body is associated with the ageing process and age-related pathologies such as cardiovascular diseases, arthritis, osteoporosis, diabetes, and cancer. While cellular senescence as a hallmark of ageing has been established, the challenge remains in identifying reliable biomarkers to detect and isolate senescent cells for further studies. One of the first steps in addressing this limitation is defining parameters for the successful generation of senescent cells <i>in vitro. </i>Our work focuses on optimizing conditions to induce senescence in mouse embryonic fibroblast (MEF) cells. Additionally, biomarkers for senescence such as senescence-associated (SA) β-galactosidase activity and lamin-b1 mRNA expression are explored to detect senescence and quantify their activity and expression levels in the senescent cells. Preliminary results using a β-galactosidase colorimetric assay reveal that the etoposide concentration most effective for generating the most senescent cells with low cell loss through apoptosis was 1 μM. Due to SA β-galactosidase being an insufficient reporter when detected through a colorimetric assay, ongoing work includes measuring SA β-galactosidase activity using fluorescence and exploring changes in lamin-b1 mRNA expression using quantitative reverse transcription polymerase chain reaction (RT-qPCR) as a potential factor that improves identification and characterization of senescent cells. Completion of this work will establish a senescence model which can be used to identify reliable senescence markers, allowing advancements in the design of novel bioanalytical strategies that track and detect senescence by mass cytometry.

Establishing an etoposide-treated mouse embryonic fibroblast (MEF) cell model for evaluation of reliable senescence markers

ACS Spring 2022

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