Abstract
Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.
Highlights
Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality
The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1
Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes
Summary
This section describes the preparation of the solid YEPD plates for use in the replicative life span experiment and the preparation of yeast cells for life span analysis. 6. Remove the cells and lightly patch cells from each coded strain to fresh YEPD plates, which will serve as the experimental plates used for the replicative life span analysis. We describe how to position the yeast cells on the plate and how to obtain virgin daughter cells for replicative life span analysis From this point on, all plates should be parafilmed, except when undergoing dissection, in order to prevent dessication. Between the 10th and 11th positions in the line, create a horizontal series of [7,8] holes in the agar This will serve as a marker to orient you on the plate during subsequent iterations of dissection and daughter cell removal. Repeat steps 2.1-2.6 for each plate in the experiment
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