Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Summary

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Abstract

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

Introduction

Budding yeast is a powerful model organism in aging research. However, a conventional lifespan assay in yeast relies on microdissection, which is not only labor intensive but also low throughput^1,2. In addition, the traditional microdissection approach does not provide a detailed view of various cellular and molecular features in the single mother cells as they age. The development of microfluidic devices has enabled an automated procedure to measure yeast lifespan as well as to follow molecular markers and various cellular phenotypes throughout the lifespan of the mother cells^3,

Protocol

1. Silicon Wafer Mold Fabrication

NOTE: The photomask is designed with AutoCAD software and manufactured by a commercial company. This design contains three layers of different patterns (Supplementary File 1). The heights of the first, second, and third layers are about 4 µm, 10 µm, and 50 µm, respectively. The silicon wafer mold was created from the photomask using soft lithography^9,10.

1. Bake a silicon wafer at 200 °C for 10 min to evaporate the water vapor. Spin coat nega....

Results

After the experiments, the lifespans of the cells and many cellular and molecular phenotypes can be extracted from the recorded time-lapse images. Since there are a number of different features that can be extracted from each cell, the first step of the analysis is to annotate the cells and events, including the positions and boundaries of the cells and the timing of various events that are being tracked, such as the budding events. These annotations will make it easier to return to the s.......

Discussion

The PDMS device needs to be freshly made. Otherwise, the air bubbles caused by inserting tubes into the device will be difficult to remove. Step 3.4 is important to improve the cell loading efficiency by concentrating the cells. To increase the throughput of the experiment, 4 to 6 modules on the same PDMS chip connected to independently operating pumps are typically used to perform 4 to 6 different experiments (different strains or media compositions) simultaneously.

Compared to the convention.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3'' <111> silicon wafer	Addison Engineering
SU-8 2000 and 3000 Series	MicroChem
Sylgard® 184 Silicone Elastomer Kit	ellsworth	2065622	Include Sylgard® silicone elastomer base and curing agent
Petri dishes	VWR	391-1502
Harris Uni-core™ punch (I.D. 0.75 mm)	Sigma-Aldrich	29002513
24 mm x 40 mm SLIP-RITE® cover glass	Thermo Fisher Scientific	102440
3M Scotch Tape	ULINE	S-10223
VWR® Razor Blades	VWR	55411-050
Pure Ethanol, Koptec	VWR	64-17-5
Whoosh-Duster™	VWR	16650-027
5 mL BD Syringe (Luer-Lock™ Tip)	Becton, Dickinson and Company	309646
PTFE Standard Wall Tubing (100 ft, AWG Size: 22, Nominal ID: 0.028)	Component Supply Company	SWTT-22
Needle Assortment	Component Supply Company	NEKIT-1
Desiccator	HACH	2238300
Lab Oven	Fisher Scientific	13246516GAQ
Nikon TE2000 microscope with 40X and 60X objective	Nikon
Zeiss Axio Observer Z1 with 40X and 60X objective	Zeiss
Syringe Pump	Longerpump	TS-1B