(1012-A) A novel single-cell fluorescence microscopy-based analysis for the detection of biomarkers of cellular senescence
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Introduction. Cellular senescence is a complex state of irreversible cell cycle arrest with a secretory phenotype associated with age-related diseases. Senescence-associated beta galactosidase (SA-β gal) is one of the hallmark biomarkers of senescent cell detection along with the increased P53 and P16 expression, and cell and nucleus areas. Traditional method of SA-β gal determination employs cytochemical or histochemical staining and manual counting of positively stained cells. This method, however, has multiple disadvantages. In addition to its laborious procedure, these approaches often assess a population of cells as a whole. As a result, given the considerable heterogeneity among cells of a population in the level of different senescence markers, these analyses do not show a robust induction of senescence. This is particularly a disadvantage for developing drugs against senescence. Aim. To develop a novel method of single-cell detection and analysis of senescence markers. Methods. To establish an accelerated model of senescence, human-derived fibroblasts were treated with the chemotherapeutic agent, Mitomycin C (MMC) (50-600 nM), or vehicle for 72 h, and further cultured in normal media for five days. IN Cell Analyzer 2200 microscope was used to detect SA-β gal (stained using a commercial fluorescent enzymatic kit), and P21 and P16 proteins (stained by antibodies). IN Carta Image Analysis Software was used to quantify senescence markers. An “induction threshold” value was set by determining the value below which 90% of values in the control no-MMC group lie. Accordingly, percent of values above threshold was assigned for each group. Results. Assessment of fluorescent intensity of SA-β gal, showed an around 2-fold increase in the average values of all cells with MMC treatment. We next plotted a histogram of values binned based on different SA-β gal fluorescence levels in cells. This histogram clearly indicated the considerable heterogeneity in SA-β gal levels. Using our “induction threshold” method, we showed an around 19-fold increase in SA-β gal levels in MMC-treated groups compared to the control. Similar trend of results was found in other biomarkers of cellular senescence including P53 and P16 expression, and cell and nucleus areas. Discussion. We developed a novel analysis method of cellular senescence biomarkers at single-cell level. This method helps in identifying sub-populations of senescent cells and provides a robust platform for senotherapeutic discovery.