A Microfluidics-Based Assay for the Discovery of Gametogenic Rejuvenation Factors in Saccharomyces cerevisiae

Saccharomyces cerevisiae progressively ages through asymmetrical cell divisions, retaining age-associated damage in the mother cell while producing young daughter cells. Gametogenesis completely resets the replicative lifespan of aged cells, thus rewinding the aging clock for the next generation. Although gametogenic rejuvenation in yeast has been described before, techniques to quantify the resetting of replicative lifespan at high throughput have been lacking. Previous methods for quantifying rejuvenation following gametogenesis were limited to low-throughput, labor-intensive approaches that relied on repeated manual micromanipulation of individual cells. Here, we describe in detail a high-throughput microfluidics-based microscopy assay that enables systematic characterization of the factors required for gametogenic rejuvenation in S. cerevisiae. This method leverages two inert genetic modifications that enable microfluidics-based assessment of gamete replicative lifespan. Upon age sorting and tetrad dissociation, conditionally sterile gametes are loaded into microfluidics-based imaging devices and identified using a gamete-specific marker. Long-term time-lapse imaging enables robust tracing and comparison of replicative lifespans of gametes derived from young and aged precursor cells. Here, we provide a step-by-step protocol that enables systematic quantification of gamete lifespan and high-throughput identification of molecular factors mediating gametogenic cellular rejuvenation in S. cerevisiae.