Summary
In this article we present a general protocol for measuring the replicative life span of yeast mother cells.
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.
Protocol
Part 1: Prepare strains and plates for replicative life span analysis 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. Using appropriate sterile technique, prepare YEPD agar plates (1% yeast extract, 2% bacto-peptone, 2% agar, 2% glucose) that will be used for culturing yeast cells and for replicative life span analysis. You should prepare at least 2 plates for …
Acknowledgements
This work was supported by a grant to M.K and B.K.K. from the Ellison Medical Foundation. M. K. is an Ellison Medical Foundation New Scholar in Aging. We would like to thank Soumya Kotireddy for assistance during filming.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Agar | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0145-17-0 (214530) | |
| Bacto-Peptone | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0118-17-0 (211677) | |
| Yeast Extract | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0886-17-0 (288620) | |
| Glucose |
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Tags
Replicative Life SpanBudding YeastAgingCellular ComponentsOrganellesMortalitySaccharomyces CerevisiaeBiology Of AgingAge-associated PhenotypesLife Span ParadigmsChronological Life SpanQuiescence-like StatePost-mitotic CellsReplicative Life Span AssayDaughter CellsSenescenceMitotically Active CellsLight Microscope
Cite This Article
Steffen, K. K., Kennedy, B. K., Kaeberlein, M. Measuring Replicative Life Span in the Budding Yeast. J. Vis. Exp. (28), e1209, doi:10.3791/1209 (2009).