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 lithography9,10.
<ol>
	<li>Bake a silicon wafer at 200 &#176;C for 10 min to evaporate the water vapor. Spin coat negative photoresist SU-8 onto the silicon wafer. 
	NOTE: Spin coat SU-8 3005 at 5,000 rpm for 30 s to generate the first layer, SU-8 2010 at 3,000 rpm for 30 s to generate the second layer, and SU-8 3025 at 2,000 rpm for 30 s to generate the third layer.</li>
	<li>Soft-bake the coated wafer before the pattern transfer. Align and expose the wafer in &#34;direct contact&#34; mode using a mask aligner. 
	NOTE: Soft-bake the wafer for 2 min at 95 &#176;C for the first layer, 3 min at 95 &#176;C for the second layer, and 15 min at 95 &#176;C for the third layer. Use the exposure dose at 100 mJ/cm2 for the first layer, 130 mJ/cm2 for the second layer, and 200 mJ/cm2 for the third layer.</li>
	<li>After exposure, post-bake the wafer and develop it using SU-8 developer. Dry the wafer using an N2 gun and hard-bake the wafer at 200 &#176;C for 30 min. 
	NOTE: The silicon wafer mold is fixed to a 15 cm-diameter plastic Petri dish using scotch tape, with the pattern side facing up. Usually, we put several identical chip structures on the same mold, which allows multiple microfluidic chips to be fabricated at the same time. Each mold can be re-used many times to fabricate microfluidic chips.</li>
</ol>
2. Microfluidic Chip Fabrication
<ol>
	<li>Place a clean weighing boat on a balance and tare the balance. Pour 50 g of PDMS base into the weigh boat.</li>
	<li>Add 5 g of PDMS curing agent to the weigh boat (w/w ratio of 1:10 to the PDMS base). 
	NOTE: This volume is based on a 15 cm-diameter Petri dish with the mold. Adjust the amount of reagent if using a Petri dish of a different size.</li>
	<li>Stir the PDMS base and the curing agent with a disposable pipette. Start from the edge of the weighing boat and slowly move inwards. Stir thoroughly for several minutes until small bubbles form throughout the mixture; thorough mixing is essential for PDMS polymerization.</li>
	<li>Pour the mixture slowly into the Petri dish; the mixture must completely cover the silicon wafer mold.</li>
	<li>Place the Petri dish in a vacuum for 10 min to remove all the air bubbles from the PDMS mixture. If bubbles remain on the surface of the mixture, use a pipette to blow them out.</li>
	<li>Incubate the silicon wafer mold with PDMS in an oven at 75 &#176;C for about 2 h.</li>
	<li>Gently peel the layer of polymerized PDMS off the silicon wafer mold, avoiding any damage to the construction of the wafer mold. Alternatively, cut the PDMS directly from the silicon wafer mold with a minimum 5-mm margin around the pattern using a single-edge industrial razor blade; gently peel the PDMS layer off the wafer mold.</li>
	<li>Place the PDMS layer on the bench with the pattern side facing up. Carefully cut out the individual chip with a single-edge industrial razor blade, retaining an adequate margin from the edge of each single chip to avoid construction damage.</li>
	<li>With the pattern side facing up, use a punch pen (0.75-mm I.D.) to punch holes straight down through the inlet and outlet circles on each side of the channels. 
	NOTE: These holes create the pathway for the flow of medium. Therefore, it is crucial to go through the circles and punch all the way through the PDMS layer. Make sure to remove the PDMS columns from the hole.</li>
	<li>Check every punched hole by inserting the punch pen needle again into the hole. Make sure the needle can come out from the other side, indicating that there is no blockage. Apply tape to the pattern surface, then gently peel off the tape to clean dust particles. Repeat this step at least three times. Leave a clean piece of scotch tape on the PDMS to maintain sterilization.</li>
	<li>Repeat this procedure on the opposite side of the PDMS and leave the last piece of tape on as well.</li>
	<li>Prepare a 24 mm x 30 mm cover glass with a thickness of 0.13-0.17 mm. Spray 70% ethanol on the glass and dry with dust remover to sterilize the surface; additionally, the glass can be washed with autoclaved water and dried with dust remover.</li>
	<li>Transfer the cover glass and PDMS to a plastic plate. Remove the scotch tape from the PDMS and place the pattern side facing up. Avoid any contact with the pattern surface during the transfer.</li>
	<li>Place the plastic plate in the plasma machine. Apply oxygen plasma treatment to the PDMS and the cover glass to render the surfaces hydrophilic with the following operation parameters: exposure, 75 s; gas stabilization, 20 cc/min; pressure, 200; and power, 100 W.</li>
	<li>Carefully place the PDMS onto the cover glass, connecting both hydrophilic surfaces (the surfaces that faced up during the plasma treatment). Make sure that there are no air bubbles between the PDMS and the cover glass.</li>
	<li>Incubate the PDMS chip in an oven at 75 &#176;C for at least 2 h. 
	&#8203;NOTE: Deficient microfluidics may cause liquid leakage onto the microscope during the experiment. Therefore, it is important to check the bond between the PDMS and the cover glass by slightly lifting the PDMS from the edges with tweezers. Gently lifting the PDMS should not separate it from the cover glass, indicating a successful bond.</li>
</ol>
3. Preparing for the Experiment
<ol>
	<li>Tube preparation.
	<ol>
		<li>Submerge the input and output tubes in 70% ethanol solution separately 1 day before PDMS chip preparation.</li>
		<li>Fill a 5-mL syringe with autoclaved water to wash the tubes. Wash each tube by plugging the tube on the syringe and flushing the tube with water.</li>
		<li>Repeat the washing step at least 3 times to clean ethanol residue.</li>
	</ol>
	</li>
	<li>Examine the PDMS channels under an optical microscope with a 10X objective to make sure the structure is consistent and intact. Stabilize the PDMS chip onto the microscope platform using scotch tape. 
	NOTE: Make multiple chips at one time in step 2 to ensure that there are backup chips, especially if making the device for the first time.</li>
	<li>Fill a 5-mL syringe with autoclaved water. Secure the syringe to a syringe pump and insert the corresponding input and output tubes. Set the washing speed to 750 &#181;L/h to rinse the chip for about 10 min. The air bubbles on the branch channels should be washed away during this period; if there are remaining bubbles, manually adjust the speed to eliminate the bubbles.</li>
	<li>Yeast sample preparation.
	<ol>
		<li>Transfer 1 mL of prepared yeast sample (OD600 0.6-0.8) into two 1.5-mL microcentrifuge tubes and centrifuge for 5 min at 3,000 x g.</li>
		<li>Remove the majority of the supernatant from each tube and combine the remainder to form a 0.5-mL sample.</li>
	</ol>
	</li>
	<li>Pause the syringe pump and remove the input tubes from the PDMS chip.</li>
	<li>Manually reverse the syringe pump to suck a small air bubble (1 to 2 cm in length) into each input tube. Move on to suck in the yeast sample; the air bubble will show the sample boundary and indicate how much sample has been drawn. 
	NOTE: Avoid sucking the sample into the syringe.</li>
	<li>Insert the input tube back into the PDMS chip and restart the pump to load the cells at a speed of 750 &#181;L/h.</li>
	<li>Leave the syringe pump on for 10 min then and examine the cell loading progression under the microscope; a successful loading will trap cells under more than 60% of suspend pillar.</li>
	<li>Switch to nutrition solution and adjust the speed to 400 &#181;L/h for cell culturing.</li>
	<li>Select observation positions for the microscope. 
	NOTE: For lifespan measurements, images were taken once every 15 min by an optical microscope with a 40x objective. For the fluorescent reporter analysis, images were taken once every 15 min by a fluorescence microscope with a 60X oil objective.</li>
</ol>

<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 authors declare that they have no competing financial interests.

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 ...

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7.6K Views.  Peking University. 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.

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

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 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.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

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

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

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

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

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 depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

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 level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

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

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 when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

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

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

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 hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, 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 remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

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 development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

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 regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

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