Name: using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells
Uploaded: 2017-03-30T15:00:00
Description: Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.

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 (<a href="http://cloudfront.jove.com/files/ftp_upload/55412/single_cell_trap.dwg" target="_blank">Supplementary File 1</a>). The heights of the first, second, and third layers are about 4 &#181;m, 10 &#181;m, and 50 &#181;m, respectively. The silicon wafer mold was created from the photomask using soft lithography9<...

<ol>
	<li>Mortimer, R. K., Johnston, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+span+of+individual+yeast+cells.">Life span of individual yeast cells.</a> Nature. 183 (4677), 1751-1752 (1959).</li><li>Polymenis, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology:+High-tech+yeast+ageing.">Cell biology: High-tech yeast ageing.</a> Nature. 486 (7401), 37-38 (2012).</li><li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phenotyping+of+aging+in+single+yeast+cells+using+a+novel+microfluidic+device.">Molecular phenotyping of aging in single yeast cells using a novel microfluidic device.</a> Aging Cell. 11 (4), 599-606 (2012).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+yeast+replicative+aging+using+a+new+generation+of+microfluidic+device.">Single cell analysis of yeast replicative aging using a new generation of microfluidic device.</a> PLoS One. 7 (11), e48275 (2012).</li><li>Chen, K. L., Crane, M. M., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+technologies+for+yeast+replicative+lifespan+studies.">Microfluidic technologies for yeast replicative lifespan studies.</a> Mech Ageing Dev. , (2016).</li><li>Lee, S. S., Avalos Vizcarra, ., Huberts, I., H, D., Lee, L. P., Heinemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+lifespan+microscopic+observation+of+budding+yeast+aging+through+a+microfluidic+dissection+platform.">Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform.</a> Proc Natl Acad Sci U S A. 109 (13), 4916-4920 (2012).</li><li>Huberts, D. H., Janssens, G. E., Lee, S. S., Vizcarra, I. A., Heinemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+high-resolution+microscopic+observation+of+replicative+aging+in+budding+yeast.">Continuous high-resolution microscopic observation of replicative aging in budding yeast.</a> J Vis Exp. (78), e50143 (2013).</li><li>Jo, M. C., Liu, W., Gu, L., Dang, W., Qin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+yeast+replicative+aging+using+a+microfluidic+system.">High-throughput analysis of yeast replicative aging using a microfluidic system.</a> Proc Natl Acad Sci U S A. 112 (30), 9364-9369 (2015).</li><li>Mata, A., Fleischman, A. J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+multi-layer+SU-8+microstructures.">Fabrication of multi-layer SU-8 microstructures.</a> Journal of Micromechanics and Microengineering. 16 (2), 276-284 (2006).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Angewandte Chemie-International Edition. 37 (5), 550-575 (1998).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+telomerase+inactivation+accelerates+aging+independently+of+telomere+length.">Early telomerase inactivation accelerates aging independently of telomere length.</a> Cell. 160 (5), 928-939 (2015).</li><li>Boy-Marcotte, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heat+shock+response+in+yeast:+differential+regulations+and+contributions+of+the+Msn2p/Msn4p+and+Hsf1p+regulons.">The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons.</a> Mol Microbiol. 33 (2), 274-283 (1999).</li><li>Yang, X., Lau, K. Y., Sevim, V., Tang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+principles+of+the+yeast+G1/S+switch.">Design principles of the yeast G1/S switch.</a> PLoS Biol. 11 (10), e1001673 (2013).</li></ol>

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...

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 throughput1,2. 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 cells3,4,5,6,7,8. After yeast cells are loaded into a microfluidic device, they can be tracked under a microscope using automatic time-lapse imaging. With the help of imaging processing tools, various cellular and molecular phenotypes can be extracted3,6,8, including lifespan, size, fluorescent reporter, cell morphology, cell cycle dynamics, etc., many of which are difficult or impossible to obtain using the traditional microdissection method. Microfluidic devices have gained prominence in yeast aging research since their successful development a few years ago3,4,6,7. Several groups have subsequently published on variations of the earlier designs5, and many yeast labs have employed microfluidic devices for their study.
In a cell culture undergoing exponential growth, the number of aged mother cells that are available for observation is miniscule. Therefore, the general design principle of the microfluidic device for lifespan measurements is to retain mother cells and to remove daughter cells. One such designs makes use of the fact that yeast undergoes asymmetric cell division. The structures in the device will trap bigger mother cells and allow smaller daughter cells to be washed away. The microfluidic chip described in this article uses a soft polydimethylsiloxane (PDMS) pad (vertical pensile columns) to trap mother cells (Figure 1). Devices of similar design have been reported previously3,4,6,7. This protocol uses a simpler procedure to fabricate microfluidic devices and a straightforward cell-loading method that is optimized for the time-lapse imaging experiments. One of the key parameters in the microfluidic device is the width of the PDMS pads used to trap mother cells. Our device uses wider pads that can keep more mother cells under each pad, including a significant fraction of fresh mother cells that can be tracked throughout their lifespan. In addition to lifespan measurements, this protocol is useful for single cell time-lapse imaging experiments when the cells need to be tracked for many generations or when an observation throughout the entire lifespan is necessary.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3&#39;&#39; &#60;111&#62; silicon wafer</td>
			<td>Addison Engineering</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2000 and 3000 Series</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>ellsworth</td>
			<td>2065622</td>
			<td>Include Sylgard&#174; silicone elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>391-1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Uni-core&#8482; punch(I.D. 0.75 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>29002513</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm x 40 mm SLIP-RITE&#174; cover glass</td>
			<td>Thermo Fisher Scientific</td>
			<td>102440</td>
			<td></td>
		</tr>
		<tr>
			<td>3M&#160; Scotch Tape</td>
			<td>ULINE</td>
			<td>S-10223</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR&#174; Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PURE ETHANOL, KOPTEC</td>
			<td>VWR</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>WHOOSH-DUSTER&#8482;</td>
			<td>VWR</td>
			<td>16650-027</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL BD Syringe (Luer-Lock&#8482;Tip)</td>
			<td>Becton, Dickinson and Company.</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028)</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>SWTT-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Assortment</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>NEKIT-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>HACH</td>
			<td>2238300</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Oven</td>
			<td>FISHER SCIENTIFIC</td>
			<td>13246516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000 microscope with 40x and 60x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Observer Z1 with 40x and 60x objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Longerpump</td>
			<td>TS-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells

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.

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...

Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

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

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.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...