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Bioengineering

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

Published: August 20th, 2013

DOI:

10.3791/50143

1Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 2Department for the Biology of Ageing, European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, 3Institute of Biochemistry, ETH Zurich, 4Department of Health Sciences and Technology, ETH Zurich, 5Institute of Molecular Systems Biology, ETH Zurich

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 μm). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

Budding yeast is an important model organism for aging research1. Until recently studying replicative aging in yeast cells was a laborious process requiring a dissection method, in which each bud was manually removed from the mother cell2,3. To solve this problem, we recently presented a novel microfluidic setup able to track individual mother cells throughout their entire lifespan4.

In our microfluidic chip, yeast cells are trapped under soft elastomer-based micropads (see Figure 1). A continuous flow of medium washes away newly formed daughter cells and provides the cells with fresh nutrie....

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1. Production and Preparation of a Silicon Wafer Mold

Microfluidic chips are created from a silicon wafer mold produced by soft lithography. These wafers can be reused many times to produce microfluidic chips. It is advisable that production of a respective wafer is performed by a group specialized in microfluidics6.

The wafer is made in a two-step photolithography process using two different layers of negative photoresist, SU-87. The bottom laye.......

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In this protocol, cells are loaded into the microfluidic chip directly from mid-exponential culture. To ascertain whether the age distribution of cells trapped in the microfluidic chip is similar to that of the culture prior to loading, cells were stained with wheat agglutinin conjugated to FITC (WGA-FITC) to visualize bud scars. As can be seen in Figure 3, the entrapment of cells under the micropads of the microfluidic chip is not biased to cells of a certain age.

Replicative.......

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The microfluidic method described here is an important novel tool in aging research as it enables simple and automated generation of yeast replicative lifespan data in combination with continuous high resolution imaging. These attributes are major improvements over the experimental possibilities of the classical dissection method, yet there are a few limitations of the method that need to be taken into account.

Note that the determined replicative lifespan can be affected by the efficiency of .......

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We would like to thank Laura Schippers for writing the first versions of the cell loading protocol and Marcus de Goffau and Guille Zampar for scoring mitochondrial morphologies.

....

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Name Company Catalog Number Comments
Name Company Catalogue number Comments
REAGENTS
DC Sylgard 184 elastomer Mavom bv 1060040 This package contains PDMS base and PDMS curing agent.
Glass Petri dishes 120/20 mm VWR International 391-2850
Cover glasses 22x40 mm CBN Labsuppliers BV 190002240
Tough-Tags Sigma-Aldrich Z359106
Aluminum foil
Plastic disposable cup
Serological pipette 5 ml VWR International 612-1245
Scotch tape VWR International 819-1460
Baysilone paste (GE Bayer silicones) Sigma-Aldrich 85403-1EA
PTFE microbore tubing, 0.012"ID x 0.030"OD Cole Parmer EW-06417-11 Referred to as thin tubing
Tygon microbore Tubing, 0.030"ID x 0.090"OD Cole Parmer EW-06418-03 Referred to as thick tubing
Scalpel VWR International 233-5334
50 ml Luer-Lok syringes BD 300137
5 ml syringes, Luer tip VWR International 613-1599
Tweezers VWR International 232-2132
20 Gauge Luer stubs Instech Solomon LS20
Syringe filters (pore size 0.20 μm) Sigma-Aldrich 16534K
Stainless steel catheter Plug, 20 ga x12 mm Instech Solomon SP20/12
Petri dishes VWR International 391-0892
EQUIPMENT
Benchtop UV-Ozone Cleaner NOVA Scan PSD-UVT
Harvard Pump 11 Elite Harvard Apparatus 70-4505
SU-8 silicon master mold (wafer) Self-made; For details contact corresponding author
Balance Sartorius corporation ED4202S
Vacuum pump KNF Neuberger N022 AN.18
Desiccator VWR International 467-2115
Hot plate VWR International 460-3267
Optional: Metal holder for cover glass (22x40 mm) Self-made; For details contact corresponding author
(Fluorescence) Microscope with 60x objective, autofocus, time-lapse abilities and preferably an automated (motorized XY control) stage Nikon Eclipse Ti-E

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