Name: Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast (Video) | JoVE Journal
Uploaded: 2013-08-20T13:00:00.000+00:00
Duration: 10 min 41 s
Description: Learn continuous microscopic observation of replicative aging in budding yeast using a microfluidic device. This method enables high-resolution tracking of single cells throughout their lifespan.

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5 minutes

We would like to thank Laura Schippers for writing the first versions of the cell loading protocol and Marcus de Goffau and Guille Zampar for scoring mitochondrial morphologies.

1. Production and Preparation of a Silicon Wafer Mold
Microfluidic chips are created from a silicon wafer mold produced by soft lithography. These wafers can be reused many times to produce microfluidic chips. It is advisable that production of a respective wafer is performed by a group specialized in microfluidics6.
The wafer is made in a two-step photolithography process using two different layers of negative photoresist, SU-87. The bottom layer is used to generate the cell trapping area (SU-8 2002; height 3-4 &mu;m), whereas the channels are made with the top layer (SU-8 2010; height 10 &mu;m). An impression of the wafer production process can be found in Huang et al8. The drawings of the microfluidic chips can be obtained from the authors as well as advice on how to obtain a respective wafer.
<ol>
	<li>Once the wafer is obtained, cut one piece of Tough-Tag into thin strips of about 0.5 mm by 3 mm with a scalpel and place them carefully on the wafer slightly on top of the channel structure between the inlet channels and the micropad array (Figure 2A). This will result in the formation of a large additional channel in each chip, which will not only be used during cell loading, but is also important for the stable operation of the microfluidic device.</li>
	<li>Cover the glass Petri dish with a double layer of aluminum foil and put the wafer in the Petri dish. The aluminum foil will prevent PDMS from running in between the wafer and the Petri dish. If PDMS does creep in between the wafer and the glass Petri dish, the wafer will become permanently fixed in the Petri dish. Although the wafer is still useable, it will not be possible to replace the glass Petri dish if it breaks and the PDMS will need to be cut out afterwards.</li>
</ol>
2. Production of Microfluidic Chips
<ol>
	<li>Place an empty plastic cup on the balance and tare the balance. Pour 40 g PDMS base into the plastic cup (for a 12 cm diameter glass Petri dish). Add PDMS curing agent with a disposable pipette in a w/w ratio of 1:10, i.e. about 4 ml.</li>
	<li>Mix the PDMS and curing agent thoroughly for several minutes. This can be done with the disposable plastic pipette that was used to pipette the curing agent. It is important that the mixture is well mixed prior to pouring it on top of the wafer. Poor mixing will cause part of the PDMS not to polymerize.</li>
	<li>Pour the PDMS on top of the wafer in the glass Petri dish.</li>
	<li>Mixing of PDMS with the curing agent will lead to bubble formation in the PDMS mixture. These bubbles need to be removed before the PDMS polymerizes. The PDMS can be degassed after pouring it on top of the wafer by placing the Petri dish in a desiccator connected to a vacuum pump. With this setup, it takes about 30 min to remove all bubbles from the mixture.</li>
	<li>Polymerize the PDMS by placing the Petri dish with the wafer on top of a hot plate at 120 &deg;C for 1 hr and then at 65 &deg;C for another hour.</li>
	<li>Remove the aluminum foil together with the wafer and polymerized PDMS from the glass Petri dish. Peel off the aluminum foil and the thin residual layer of polymerized PDMS from the back of the wafer. The layer of PDMS on top of the wafer can then be separated from the wafer by lifting it up carefully.</li>
	<li>Place the PDMS layer upside down (with the channels facing up) on the bench and cut out the single chip designs imprinted on the PDMS carefully with a scalpel. Try to retain about 3 mm of PDMS around the edges of the channels to improve attachment of the chip to the cover glass later on.</li>
	<li>Punch holes in the PDMS at the ends of the inlet, outlet and side channel (Figure 2A) by pushing a 20 Gauge Luer stub all the way through the PDMS in a straight manner.</li>
</ol>
Make sure the PDMS chip is still lying upside down on the bench while punching the holes. This prevents PDMS from sticking to the inside of the channel, which could cause blockage of the channel.
<ol start="9">
	<li>After punching each hole, remove the column of PDMS in the Luer stub with tweezers before pulling the stub out again. The PDMS column could otherwise stay behind and block the just punched hole.</li>
	<li>Clean the surfaces of the chip from dust particles and residual PDMS by placing scotch tape on it and immediately removing the tape again. Similarly clean the cover glass to which the chip will be attached.</li>
	<li>Place the cleaned chip and cover glass below the UV lamp of the Benchtop UV-Ozone Cleaner. The sides that need to be bonded together must face the lamp.</li>
	<li>Expose the PDMS and cover glass to the UV light for 6-8 min and directly afterwards put the chip on the cover glass by placing the exposed surfaces on top of each other.</li>
	<li>Gently tap around the sides of the chip to promote attachment of the PDMS to the cover glass and to remove any air bubbles. Do not tap on top of the channel structure as this may cause the micropads to get attached to the cover glass as well.</li>
	<li>Place the newly made microfluidic setup on a hot plate at 100 &deg;C for 60 min. Test the strength of the bonding between the cover glass and PDMS chip by trying to lift up the edges of the PDMS chip slightly. When the PDMS block cannot be lifted from the glass surface, the bonding is successful and the chip is ready for use.</li>
</ol>
3. Preparing the Chip for Cell Loading
Failure of a microfluidic device can potentially cause leakage of medium into the microscope. To avoid this, it is advisory to use a metal holder and/or seal the sides of the chip where the PDMS meets the glass with nail polish or epoxy glue. This prevents leakage of medium in case the PDMS chip is not bonded well enough to the cover glass. Also the tubing connections can be secured around the insertion points in a similar manner to avoid leakage of medium. A home-made water sensor that switches off the syringe pump in case of leakage can be used as an extra safety precaution. Drawings of the metal holder especially designed for the microfluidic chip can be obtained from the authors.
<ol>
	<li>Place the microfluidic chip in the metal holder, with silicone gel between the glass of the chip and the metal parts of the holder to create a waterproof seal. Screw the nuts gently, not to break the glass.</li>
	<li>Connect thin tubes (ca. 5-15 cm long) to the side channel and the outlet channel of the chip. The use of tweezers makes it easier to insert the tubing in the punched holes.</li>
	<li>Fill a 50 ml Luer-Lok syringe with culture medium, remove the air from the syringe and connect it sequentially to a syringe filter, a 20 Gauge Luer stub, a short thick tube (ca. 2 cm long) and a thin tube. Make sure that the thin tube is long enough to span the distance between the syringe pump and the microscope stage.</li>
	<li>Place the syringe in the syringe pump, fast forward the pump until the thin tube is completely filled with medium.</li>
	<li>Push the thin tube connected to the syringe into the inlet channel of the chip and let the medium flow through the chip at 10 &mu;l/min (Figure 2B). Collect the medium leaving the chip (e.g. in a Petri dish). The medium will run out via the side channel because of the resistance difference between the Tough-Tag made large side channel and the outlet channel.</li>
	<li>Place the chip in the microscope stage and set the focus of the microscope on the pads.</li>
</ol>
The use of oil immersion objectives is preferred over that of water immersion objectives because oil will not dry out over the time-course of the experiment.
Depending on the microscope there might be issues with retaining the focus during the initial hours of the experiment. It is therefore advisable to leave the chip for one to two hours in the microscope stage so it can settle down before loading cells and starting the experiment.
4. Loading of Yeast Cells into the Microfluidic Chip
<ol>
	<li>Connect a 5 ml Luer tip syringe to a 20 Gauge Luer stub and a thick tube (ca. 3 cm).</li>
	<li>Take a sample of the cell culture to be loaded into the chip.</li>
</ol>
The preferred cell count for loading is between 1-5 x 106 cells per ml. Cultures with higher cell counts need to be diluted before loading. When culturing cells on YPD, cell loading efficiency can be improved many-fold if cells are first washed with and resuspended in minimal medium containing a similar glucose percentage before loading. During operation of the chip, YPD medium can again be safely used.
<ol start="3">
	<li>Load approximately 1 ml of the cell suspension into the syringe and remove all the air from the syringe.</li>
	<li>Decrease the flow rate of the syringe pump to 0.5 &mu;l/min. The low and continued flow enforced by the syringe pump during loading (in step 4.5) will push non-attached cells out via the side channel (Figure 2C).</li>
	<li>Connect the syringe containing the cell suspension to the tube of the outlet channel. Load the cells by pressing on the plunger of the syringe gently. Observe the cells coming in and settling underneath the micropads via the ocular or via the computer screen.</li>
	<li>Maintain pressure on the plunger until sufficient cells settle underneath the pads. The optimum load is 1-3 cells per pad. As cells settle underneath the pads randomly, it is quite common that a few pads do not contain any cells and other pads are completely filled with cells.</li>
	<li>Disconnect the syringe used for cell loading and the thick tube connected to it from the thin tubing of the outlet channel, flush the side channel for a couple of minutes at higher flow rates (10 &mu;l/min) to remove air bubbles and/or cells that did not yet exit the chip (Figure 2B). If cells are not removed from the side channel, they can start to grow in the side channel and interfere with the experiment later on.</li>
	<li>Put the flow of the syringe pump back down to 0.5 &mu;l/min and close the side channel off by connecting it to a thick tube (ca. 5 cm long) containing a catheter plug at its end (Figure 2D).</li>
	<li>Increase the flow to a final flow rate of 1-5 &mu;l/min. Flow rates may vary depending on medium and yeast strain.</li>
	<li>Start a movie with the microscope and camera settings suitable for the goal of the experiment. Make sure that the speed of the microscope stage is set to such a low speed that the oil can follow. For determination of replicative lifespan, image cells every 10 min. To protect cells from the minute amounts of UV light emitted by the halogen lamp, which is used during DIC acquisition, it is advisable to place an additional UV filter in the light path. When using fluorescence microscopy, it is preferred to image the cells less frequently (e.g. every 20 min) to avoid phototoxic effects. The experiment is completed when all cells loaded at the start of the experiment are dead, which under normal conditions takes up to 5 days. It is recommended to regularly check the focus of the images during the experiment and if necessary adjust it.</li>
</ol>

<ol><li>Kaeberlein, M., McVey, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+yeast+to+discover+the+fountain+of+youth%5BTitle%5D)+AND+%22Sci.+Aging+Knowledge+Environ%22%5BJournal%5D)">Using yeast to discover the fountain of youth.</a> Sci. Aging Knowledge Environ. 2001 (1), pe1(2001).</li>
<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>Steffen, K. K., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+replicative+life+span+in+the+budding+yeast%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Measuring replicative life span in the budding yeast.</a> J. Vis. Exp. (28), e1209(2009).</li>
<li>Lee, S. S., Avalos Vizcarra, I., Huberts, D. H., 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>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. , (2012).</li>
<li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Soft+Lithography%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition%22%5BJournal%5D)">Soft Lithography.</a> Angewandte Chemie International Edition. 37 (5), 550-575 (1998).</li>
<li>Mata, A., Fleischman, A. J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+of+multi-layer+SU-8+microstructures%5BTitle%5D)+AND+%22Journal+of+Micromechanics+and+Microengineering%22%5BJournal%5D)">Fabrication of multi-layer SU-8 microstructures.</a> Journal of Micromechanics and Microengineering. 16 (2), 276-284 (2006).</li>
<li>Huang, Y., Agrawal, B., Clark, P. A., Williams, J. C., Kuo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+cancer+stem+cell+migration+using+compartmentalizing+microfluidic+devices+and+live+cell+imaging%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging.</a> J. Vis. Exp. (58), e3297(2011).</li>
<li>Kaeberlein, M., Kirkland, K. T., Fields, S., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+determining+yeast+replicative+life+span+in+a+long-lived+genetic+background%5BTitle%5D)+AND+%22Mechanisms+of+Ageing+and+Development%22%5BJournal%5D)">Genes determining yeast replicative life span in a long-lived genetic background.</a> Mechanisms of Ageing and Development. 126 (4), 491-504 (2005).</li>
<li>Scheckhuber, C. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reducing+mitochondrial+fission+results+in+increased+life+span+and+fitness+of+two+fungal+ageing+models%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models.</a> Nat. Cell Biol. 9 (1), 99-105 (2007).</li>
<li>Defossez, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elimination+of+replication+block+protein+Fob1+extends+the+life+span+of+yeast+mother+cells%5BTitle%5D)+AND+%22Mol.+Cell%22%5BJournal%5D)">Elimination of replication block protein Fob1 extends the life span of yeast mother cells.</a> Mol. Cell. 3 (4), 447-455 (1999).</li>
<li>Kaeberlein, M., McVey, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+SIR2%2F3%2F4+complex+and+SIR2+alone+promote+longevity+in+Saccharomyces+cerevisiae+by+two+different+mechanisms%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms.</a> Genes Dev. 13 (19), 2570-2580 (1999).</li>
<li>Shcheprova, Z., Baldi, S., Frei, S. B., Gonnet, G., Barral, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mechanism+for+asymmetric+segregation+of+age+during+yeast+budding%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A mechanism for asymmetric segregation of age during yeast budding.</a> Nature. 454 (7205), 728-734 (2008).</li>
<li>Vanoni, M., Vai, M., Popolo, L., Alberghina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+heterogeneity+in+populations+of+the+budding+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.</a> J. Bacteriol. 156 (3), 1282-1291 (1983).</li>
<li>Huh, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+analysis+of+protein+localization+in+budding+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Global analysis of protein localization in budding yeast.</a> Nature. 425 (6959), 686-691 (2003).</li>
<li>Zhang, Y., Luo, C., Zou, K., Xie, Z., Brandman, O., Ouyang, Q., Li, H. <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>
</ol>

The authors declare that they have no competing financial interests.

The microfluidic method described here is an important novel tool in aging research as it enables simple and automated generation of yeast replicative lifespan data in combination with continuous high resolution imaging. These attributes are major improvements over the experimental possibilities of the classical dissection method, yet there are a few limitations of the method that need to be taken into account.
Note that the determined replicative lifespan can be affected by the efficiency of ...

Budding yeast is an important model organism for aging research1. Until recently studying replicative aging in yeast cells was a laborious process requiring a dissection method, in which each bud was manually removed from the mother cell2,3. To solve this problem, we recently presented a novel microfluidic setup able to track individual mother cells throughout their entire lifespan4.
In our microfluidic chip, yeast cells are trapped under soft elastomer-based micropads (see Figure 1). A continuous flow of medium washes away newly formed daughter cells and provides the cells with fresh nutrients. In a single experiment, up to 50 mother cells can be monitored in a fully automated manner throughout their entire replicative lifespan. Due to the excellent optical properties of the microfluidic chip, it is possible to simultaneously monitor different aspects of yeast cell biology (e.g. by using fluorescent proteins).
 
Compared to the classical dissection method, the microfluidic setup provides substantial advantages. It ensures a defined and constant environment during the whole aging experiment. It requires no expensive specialized equipment and can be run on any microscope equipped with automated focus and time-lapse abilities as well as temperature-control for cell cultivation. The production and operation of the microfluidic chips can be learned within a few days. In addition, cells can be directly loaded from an exponentially growing culture, an advantage over another recently published microfluidic method5, which requires biotinylation of mother cells.a Combined with high-resolution imaging, the here described method can be used to measure gradual changes in cellular morphology, protein expression and localization during yeast aging in an unprecedented manner. The capability for long-term monitoring of single cells also provides unique possibilities for yeast cell cycle studies.
aThis method has recently been optimized to remove the biotinylation from the protocol16, which was published while this manuscript was in review.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>DC Sylgard 184 elastomer </td><td>Mavom bv</td><td>1060040</td><td>This package contains PDMS base and PDMS curing agent.</td></tr><tr><td>Glass petri dishes 120/20 mm </td><td>VWR International</td><td>391-2850</td><td></td></tr><tr><td>Cover glasses 22x40 mm </td><td>CBN Labsuppliers BV</td><td>190002240</td><td></td></tr><tr><td>Tough-Tags </td><td>Sigma-Aldrich</td><td>Z359106</td><td></td></tr><tr><td>Aluminum foil </td><td></td><td></td><td></td></tr><tr><td>Plastic disposable cup </td><td></td><td></td><td></td></tr><tr><td>Serological pipette 5 ml </td><td>VWR International</td><td>612-1245</td><td></td></tr><tr><td>Scotch tape </td><td>VWR International</td><td>819-1460</td><td></td></tr><tr><td>Baysilone paste (GE Bayer silicones) </td><td>Sigma-Aldrich</td><td>85403-1EA</td><td></td></tr><tr><td>PTFE microbore tubing, 0.012&quot;ID x 0.030&quot;OD </td><td>Cole Parmer</td><td>EW-06417-11</td><td>Referred to as thin tubing </td></tr><tr><td>Tygon microbore Tubing, 0.030&quot;ID x 0.090&quot;OD </td><td>Cole Parmer</td><td>EW-06418-03</td><td>Referred to as thick tubing </td></tr><tr><td>Scalpel </td><td>VWR International</td><td>233-5334</td><td></td></tr><tr><td>50 ml Luer-Lok syringes </td><td>BD</td><td>300137</td><td></td></tr><tr><td>5 ml syringes, Luer tip </td><td>VWR International</td><td>613-1599</td><td></td></tr><tr><td>Tweezers </td><td>VWR International</td><td>232-2132</td><td></td></tr><tr><td>20 Gauge Luer stubs </td><td>Instech Solomon</td><td>LS20</td><td></td></tr><tr><td>Syringe filters (pore size 0.20 &amp;mu;m) </td><td>Sigma-Aldrich</td><td>16534K</td><td></td></tr><tr><td>Stainless steel catheter Plug, 20 ga x12 mm </td><td>Instech Solomon</td><td>SP20/12</td><td></td></tr><tr><td>Petri dishes </td><td>VWR International</td><td>391-0892</td><td></td></tr><tr><td colspan="4"> &lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Benchtop UV-Ozone Cleaner </td><td>NOVA Scan</td><td>PSD-UVT</td><td></td></tr><tr><td>Harvard Pump 11 Elite </td><td>Harvard Apparatus</td><td>70-4505</td><td></td></tr><tr><td>SU-8 silicon master mold (wafer) </td><td></td><td></td><td>Self-made; For details contact corresponding author </td></tr><tr><td>Balance </td><td>Sartorius corporation</td><td>ED4202S</td><td></td></tr><tr><td>Vacuum pump </td><td>KNF Neuberger</td><td>N022 AN.18</td><td></td></tr><tr><td>Desiccator </td><td>VWR International</td><td>467-2115</td><td></td></tr><tr><td>Hot plate </td><td>VWR International</td><td>460-3267</td><td></td></tr><tr><td>Optional: Metal holder for cover glass (22x40 mm) </td><td></td><td></td><td>Self-made; For details contact corresponding author </td></tr><tr><td>(Fluorescence) Microscope with 60x objective, autofocus, time-lapse abilities and preferably an automated (motorized XY control) stage </td><td>Nikon</td><td>Eclipse Ti-E</td><td></td></tr></tbody></table>

continuous high-resolution microscopic observation of replicative aging in budding yeast

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

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

In this protocol, cells are loaded into the microfluidic chip directly from mid-exponential culture. To ascertain whether the age distribution of cells trapped in the microfluidic chip is similar to that of the culture prior to loading, cells were stained with wheat agglutinin conjugated to FITC (WGA-FITC) to visualize bud scars. As can be seen in Figure 3, the entrapment of cells under the micropads of the microfluidic chip is not biased to cells of a certain age.
Replicative...

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.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

The overall goal of this procedure is to track single haplo yeast cells during their entire replicative lifespan. The first step is to make a microfluidic chip containing an array of micro pads. Next, the yeast cells are loaded into the microfluidic chip, and because the area underneath the micro pads has a height similar to the diameter of a yeast cell, the cells will settle there.Subsequently, a continuous flow of fresh medium is applied over the trapped yeast cells, and the emerging daughter cells are washed away by the flow of medium brightfield and fluorescence. Microscopy is used to visualize changes in cell or organelle morphology, protein expression, and protein localization that may occur in aging yeast cells. The main advantage of this technique over the classical microdissection method is that it's less labor intensive and that it enables long-term continuous microscopic images of the entire lifespan of individual yeast cells.Though this method can provide insight into replicative aging, it can also be used for studies requiring continuous microscopic observation over prolonged periods of time. Generally, individuals new to this method will struggle because care needs to be taken at particular steps of the production of the micro feic chip. In addition, loading of the cells into the micro feic chip may require some experience.The silicon wafer mold is produced by soft lithography. For details of its production, please refer to the accompanying protocol. Text cut a piece of tough tag into thin strips of about 0.5 millimeters by three millimeters.With a scalpel, place the strips carefully on the silicon wafer mold slightly on top of the channel structure between the inlet channels and the micro padre. Next, cover a glass petri dish with a double layer of aluminum foil and put the wafer in the Petri dish. The aluminum foil will prevent PDMS from running in between the wafer and the Petri dish during the production of the microfluidic chips.To begin the procedure for making microfluidic chips, place an empty plastic cup on the balance and tear the balance. Pour 40 grams of PDMS base into the plastic cup. Add PDMS curing agent in a weight to weight ratio of one to 10, which in this case is about four milliliters.Using the disposable plastic pipette mixed thoroughly for several minutes, it is important that the mixture is well mixed to ensure proper polymerization of the PDMS later. Pour the PDMS on top of the wafer in the glass Petri dish. The PDMS mixture in the Petri dish will contain a lot of bubbles to Degas.The PDMS place, the Petri dish in a desiccate connected to a vacuum pump for about 30 minutes when all bubbles have been removed. Polymerize the PDMS by placing the Petri dish with the wafer on top of a hot plate at 120 degrees Celsius for one hour, followed by 65 degrees Celsius for another hour. After the two hours, remove the aluminum foil wafer and polymerized PDMS from the glass Petri dish.Peel off the aluminum foil into PDMS from the back of the wafer. Then carefully lift the layer of PDMS from the top of the wafer. Place the PDMS layer upside down with the channels facing up a the bench and carefully cut out the single chip designs imprinted on the PDMS with the scalpel.Try to retain about three millimeters of PDMS around the edges of the channels. The next step is to punch holes in the PDMS at the ends of the inlet outlet and side channel as illustrated in this diagram. To punch a hole, push a 20 gauge lure stub all the way through the PDMS in a straight manner.Remove the column of PDMS in the lure stub before pulling the stub out. Again, this prevents the PDMS column from blocking the newly punched hole. Once all the holes have been punched, clean the surfaces of the chip from dust particles and residual PDMS by placing scotch tape on it and immediately removing the tape.Similarly, clean the cover glass to which the chip will be attached. Place the chip and cover glass below the UV lamp of a benchtop UV ozone cleaner with the sides that need to be bonded together, facing the lamp exposed to UV light for six to eight minutes, and put the exposed surfaces on top of each other. Gently tap around the sides of the chip to promote attachment of the PDMS to the cover glass, and to remove any air bubbles.Do not tap on top of the channel structure as this may cause the micro pads to get attached to the cover. Glass as well. Place the newly made microfluidic setup on a hot plate at 100 degrees Celsius for one hour.Afterwards, check the bonding of the cover glass to the PDMS chip by slightly lifting up the edges of the PDMS chip. If this is not possible, the bonding is successful and the chip is ready for use. To prepare the microfluidic chip for cell loading, place the chip in the metal holder with silicone gel between the glass of the chip and the metal parts of the holder.To create a waterproof seal, screw the nuts gently to avoid breaking the glass. Connect thin tubes to the side channel and the outlet channel of the chip. The use of tweezers makes it easier to insert the tubing in the punched holes.Fill a 50 milliliter lure lock syringe with culture medium. Remove the air from the syringe and connect it sequentially to a syringe. Filter a 20 gauge lure stub, a short, thick tube and a thin tube.Place the syringe in the syringe pump and fast forward the pump until the thin tube is completely filled with medium. Push the thin tube connected to the syringe into the inlet channel of the and let the medium flow through the chip at a rate of 10 microliters per minute. Collect the medium leaving the chip in a Petri dish.The medium will run out via the side channel. Because of the resistance difference between the large tough tag made side channel and the outlet channel. Place the chip on the microscope stage and set the focus of the microscope on the pads.To begin this procedure, connect to five milliliter lure tip syringe to a 20 gauge lure stub and a thick tube. Load the syringe with approximately one milliliter of the cell suspension to be loaded into the chip. The preferred cell count for loading is between one and five times 10 of the six cells per milliliter.Decrease the flow rate of the syringe pump to 0.5 microliters per minute. The most difficult part of this procedure is the loading of the cells into the microme chip. This usually requires some practice.Connect the syringe containing the cell suspension to the tube of the outlet channel. Load the cells by pressing on the plunger of the syringe. Gently observe the cells coming in and settling underneath the micro pads via the ocular or the computer screen.Maintain pressure on the plunger until sufficient cells settle underneath the pads. The optimum load is one to three cells per pad. Disconnect the syringe used for cell loading and flush the side channel for a couple of minutes at higher flow rates to remove air bubbles and or cells that did not yet exit the chip.Decrease the flow of the syringe pump to 0.5 microliters per minute again, and close the side channel off by connecting it to a thick tube containing a catheter plug at its end. Increase the flow of the syringe pump to a final flow rate of one to five microliters per minute. Flow rates may vary depending on the medium and yeast strain.Start a movie with the microscope and camera setting suitable for the goal of the experiment. This is an example time-lapse movie of a single wild type E cell growing on YPD. Medium images were taken every 10 minutes and the scale bar just shown represents five micrometers.The cell produces a total of 30 buds before it dies. Replicative lifespan can be determined by counting the number of buds produced by a single mother cell. This data is transformed into a lifespan curve by plotting the percentage of viable cells against the number of buds produced or the number of generations.This figure shows an example of lifespan curves obtained for a wild type yeast strain and two mutant strains. Deletion of the SER two gene results in a shorter lifespan while deletion of the FOB one gene results in a longer lifespan. Mitochondrial morphology as a function of age can be studied using the microfluidic device.An aging experiment was performed with BY 7 42 wild type yeast cells expressing ILV 3G FP, which is targeted to the mitochondria. In this figure, A representative example of age associated changes in mitochondrial morphology in a single cell is indicated by the white arrow. All images are scaled identically and the scale bar represents five micrometers.The second time lapse movie is of a single cell expressing ILV 3G FP from the experiment where mitochondrial morphology as a function of age was observed. Images were taken every 30 minutes and the scale bar represents five micrometers. This final figure shows the mitochondrial morphologies observed in the same set of cells at different replicative ages before producing their first bud after 10 buds and prior to death.An example image of each morphology class is included tubular, fragmented tubular, and spots and large spots. The morphology changes seen in mid aged cells resemble those reported previously By continuously monitoring cells as they divide. This method can help answer key questions in yeast replicative aging, such as why does a yeast cell age, what phenotypic changes are taking place, and how do these influence the replicative lifespan of the yeast?After watching this video, you should know how to create your own micro chips and study replicative aging in cells using basically any fluorescent microscope.

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Learn continuous microscopic observation of replicative aging in budding yeast using a microfluidic device. This method enables high-resolution tracking of single cells throughout their lifespan.

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

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