Name: Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells (Video) | JoVE Journal
Uploaded: 2017-03-30T15:00:00.000+00:00
Duration: 9 min 18 s
Description: Learn to fabricate microfluidic devices for measuring lifespan and cellular phenotypes in single budding yeast cells. This method enables precise tracking of mother cells and molecular markers to study aging mechanisms.

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5 minutes

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/pubmed?term=((Life+span+of+individual+yeast+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Cell+biology%3A+High-tech+yeast+ageing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Molecular+phenotyping+of+aging+in+single+yeast+cells+using+a+novel+microfluidic+device%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">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/pubmed?term=((Single+cell+analysis+of+yeast+replicative+aging+using+a+new+generation+of+microfluidic+device%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+technologies+for+yeast+replicative+lifespan+studies%5BTitle%5D)+AND+%22Mech+Ageing+Dev%22%5BJournal%5D)">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/pubmed?term=((Whole+lifespan+microscopic+observation+of+budding+yeast+aging+through+a+microfluidic+dissection+platform%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Continuous+high-resolution+microscopic+observation+of+replicative+aging+in+budding+yeast%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((High-throughput+analysis+of+yeast+replicative+aging+using+a+microfluidic+system%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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://iopscience.iop.org/article/10.1088/0960-1317/16/2/012/meta">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.annualreviews.org/doi/abs/10.1146/annurev.matsci.28.1.153">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/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">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/pubmed?term=((Early+telomerase+inactivation+accelerates+aging+independently+of+telomere+length%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((The+heat+shock+response+in+yeast%3A+differential+regulations+and+contributions+of+the+Msn2p%2FMsn4p+and+Hsf1p+regulons%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Design+principles+of+the+yeast+G1%2FS+switch%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">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><tbody><tr><td>3&#039;&#039; &amp;lt;111&gt; 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&amp;reg; 184 Silicone Elastomer Kit</td><td>ellsworth</td><td>2065622</td><td>Include Sylgard&amp;reg; 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&amp;trade; 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&amp;reg; cover glass</td><td>Thermo Fisher Scientific</td><td>102440</td><td></td></tr><tr><td>3M Scotch Tape</td><td>ULINE</td><td>S-10223</td><td></td></tr><tr><td>VWR&amp;reg; 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&amp;trade;</td><td>VWR</td><td>16650-027</td><td></td></tr><tr><td>5 mL BD Syringe (Luer-Lock&amp;trade; Tip)</td><td>Becton, Dickinson and Company</td><td>309646</td><td></td></tr><tr><td>PTFE Standard Wall Tubing (100 ft, 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

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...

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 ...

The overall goal of this experiment is to make a specialized microfluidic device and use it to measure the lifespan and cellular phenotypes of a single yeast cell. This method can help answer some of the key questions in the yeast aging field, such as why mother cells age and die and what are the earlier molecular events that drive the loss of cellular homeostasis and eventually cell aging and death? The major advantage of this technique is that we can track mother cells, track their lifespan, and the various molecular markers at the same time, so we can look for molecular events that correlate with lifespan.Though this method can provide insight into yeast aging it can also be applied to other areas, such as monitoring secondary responses to participation that requires cell checking for many generations. Generally, individuals new to this method will struggle because microfluidic device fabrication, cell loading, microscope set up require a lot of practice to perform successfully. Prior demonstration of this method is critical as it is difficult to properly load the microfluidic device with yeast.To begin fabricate a silicon wafer mold for the device using standard techniques as described in the accompanying text protocol. Place it feature side up into a 15 centimeter Petri dish. Secure the mold on the Petri dish with tapes.Next place a clean weighing boat on a balance and tear the balance. Pour 50 grams of PDMS base into the weigh boat and then add five grams of PDMS curing agent. Stir the PDMS base and curing agent with a disposable pipette.Start from the edge of the weigh boat and slowly move inwards. Stir the mixture thoroughly for several minutes until small bubbles form throughout the mixture. Pour the mixture slowly into the Petri dish, so it completely covers the silicon wafer mold.Then place the Petri dish in a vacuum for 10 minutes to remove all air bubbles from the PDMS mixture. If bubbles remain on the surface of the mixture once removed from the vacuum use a pipette to blow them out. Next, bake the device at 75 degrees Celsius for two hours.When cooled back to room temperature, carefully cut through the PDMS around the mold and then gentle peel the layer of polymerized PDMS off the silicon wafer mold, avoiding any damage to the construction of the wafer mold. 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.With the pattern side facing up, use a punch pen to punch holes straight down through the inlet and outlet circles on each side of the channels. Check each 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 are no blockages.Next use the tape to clean dust particles and debris from the top surface. Repeat this step at least three times, and leave a clean piece of Scotch tape on the PDMS to maintain a dust free surface. Then turn the device over and repeat this procedure on the opposite site of the PDMS.Leave the last piece of tape on this side as well. Now prepare a glass surface for the base of the device. Select the 24 millimeter by 30 millimeter piece of cover glass with a thickness between 13 and 17 millimeters.Spray 70%ethanol on the glass, and dry it with dust remover to sterilize the surface. Then transfer the cover glass and PDMS to a plastic plate. Remove the Scotch tape from the PDMS, and place the pattern side facing up in the plate, without contacting the top surface.Then transfer the plate into a plasma chamber. Apply an oxygen plasma treatment to the PDMS and the cover glass to render the surfaces hydrophilic. Then remove the plate from the plasma chamber and carefully connect the plasma treated surfaces of the PDMS in cover glass.Ensure that there are no air bubbles between the PDMS and the cover glass. Finally, transfer the device to an oven and bake at 75 degrees Celsius for at least two hours. Submerge the input and output tubes in 70%ethanol solution one day before PDMS chip preparation.The next day fill a five milliliter syringe with autoclaved water, and wash each tube by placing the tube on the syringe and flushing the tube with water. Repeat the washing step at least three times to remove residual ethanol. Next examine the PDMS channels under an optical microscope.Using a 10 X objective make sure that the structures are consistent and intact. Stabilize the intact PDMS chip on the microscope platform using Scotch tape. Secure a five milliliter syringe loaded with sterile water to a syringe pump and insert the corresponding input and output tubes to the device.Set the flow rate to 750 microliters per hour and rinse the chip for about 10 minutes. Watch the channels to ensure that any air bubbles are washed away. If any bubbles remain following the 10 minute rinse manually adjust the speed to eliminate the bubbles.Transfer one milliliter of a prepared yeast sample into two 1.5 milliliter microcentrifuge tubes and centrifuge the samples for five minutes at 3, 000 times G.Remove the majority of the supernatant from each tube and combine the remainder to form a 5 milliliter sample. With the yeast sample now ready, remove the input tubes from the PDMS chip. Manually reverse the syringe pump to suck in a small air bubble that is one to two centimeters in length, each input tube, and then suck in cell cultures.Then insert the input tube back into the PDMS chip and restart the pump to load the cells at a speed of 750 microliters per hour. Leave the syringe pump on for 10 minutes and then examine the cell loading progression under the microscope. A successful loading will trap cells under more than 60%of suspend pillar.To maintain the yeast cells, switch the solution in the syringe to a nutrition solution, and adjust the speed to 400 microliters per hour. The lifespan phenotype of yeast can be obtained by counting the number of buds produced by each trapped mother cell, and estimating the Kaplan-Meier estimator. Using this protocol the device frequently traps fresh daughter cells that budded off from the already trapped cells.These cells then turn into fresh mother cells which can be identified using downstream image analysis software. These fresh mother cells provide a more accurate lifespan measurement. In this experiment, the average lifespan of the fresh mother cells was slightly longer by about two generations.In addition to the number of buds, the time interval between two successive budding events can be extracted from the imaging data. Budding slows down dramatically towards the end of their lifespan indicating the unhealthy state of these aged cells. While attempting this procedure, it's important to remember to both use a freshly made chip to help avoid bubbles, and to increase the cell concentration in order to improve cell loading efficiency when needed.After its development this technique paved the way for researchers in yeast aging field to explore the early molecular changes that are causal to the aging phenotype. After watching this video you should have a good understanding of how to make this microfluidic device and how to use it to measure lifespan and cellular phenotypes in single cells.

using-microfluidic-devices-to-measure-lifespan-cellular-phenotypes

Research

JoVE Journal

Biology

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase.  Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

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

Measuring Replicative Life Span in the Budding Yeast

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

A Microfluidic Device for Studying Multiple Distinct Strains

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.

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

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.

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

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Single-cell Microfluidic Analysis of Bacillus subtilis

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Learn to fabricate microfluidic devices for measuring lifespan and cellular phenotypes in single budding yeast cells. This method enables precise tracking of mother cells and molecular markers to study aging mechanisms.

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

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