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

Summary

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.

Abstract

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.

Introduction

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 cell^2,3. 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....

Protocol

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 layer is used to generate the cell trapping area (SU-8 2002; height 3-4 μm), whereas the channels are made with the top layer (SU-8 2010; height 10 &mu....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog 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