Name: A Microfluidic Device for Studying Multiple Distinct Strains (Video) | JoVE Journal
Uploaded: 2012-11-09T12:00:00.000+00:00
Duration: 8 min 15 s
Description: Learn to image single yeast cells from different strains under identical dynamic conditions using a two-layer microfluidic device. This method prevents cross-contamination while enabling simultaneous observation of multiple strains.

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YG is supported by a fellowship from IDB. IN is an Alon fellow and a faculty fellow of the Edmond J&#x202D;. &#x202C;Safra Center for Bioinformatics at Tel Aviv University&#x202D;.&#x202C; This research was supported by ISF grant 1499/10.

1. Creating a Scotch-tape Master5
<ol>
	<li>Draw or print out desired microchannel layout, to scale, on paper. In our case the design consists of two Y-shaped channels, each 3 mm wide (Figure 2).</li>
	<li>Cover a glass-slide with layers of scotch tape. The number of layers will determine the height of the channel (about 60 &mu;m per layer). We used 3 scotch tape layers.</li>
	<li>Place the layout design on a flat surface. Align the slide over the design pattern. Carefully cut the tape on the glass slide with a scalpel according to the layout.</li>
	<li>Remove the Scotch tape from all regions of the glass slide except those in the layout of the microchannel.</li>
	<li>Place the slide in a heating oven at 65 &deg;C for 2-3 min. Clean gently with ethanol.</li>
</ol>
2. Fabricating a PDMS Microfluidic Device
<ol>
	<li>Mix the base and curing components of PDMS as recommended by the manufacturer. We use a 10:1 ratio of base to curing agent. Pour about 30 ml of PDMS mixture into a Petri dish to a height of ~0.5 cm. Degas the PDMS in vacuum if needed. Submerge the glass slide in the PDMS with the patterned Scotch tape facing up. Placing the pattern after the PDMS prevents formation of air bubbles between the slide and dish bottom. Cure for 48 hr at 65 &deg;C, making sure the dish is horizontal using a bubble level.</li>
	<li>In a new 90 mm Petri dish pour 3 ml of PDMS mix, resulting in a layer about 0.5 mm deep. (If a spin coater is available, it can be used for this stage).</li>
	<li>Degas and cure as in 2.1.</li>
	<li>Gently cut out the microfluidic device to the desired size with a scalpel. This layer will be termed the "flow layer".</li>
	<li>Cut out a similarly sized piece of thin layer PDMS from the second Petri dish. This will be termed the "wells layer".</li>
	<li>Use an appropriately sized biopsy puncher (We use 1.2 mm ID) to punch holes for the inlets and outlets in the flow layer.</li>
	<li>Place the wells layer on a thick glass slide over the design layout and punch out wells, using 2 mm ID biopsy puncher, in alignment with the microchannels on the layout.</li>
	<li>Clean both PDMS layers as well as a 24 mm x 60 mm glass coverslip with ethanol. Air-dry and keep clean.</li>
	<li>Plasma treat both the glass coverslip and the wells layer PDMS using either standard plasma etcher (for non-reversible bonding) or a hand-held corona treater6 for reversible bonding. Carefully place the wells layer on top of the coverslip to cause adhesion (Figure 3). Gently rub out any air bubbles (if needed).</li>
</ol>
3. Cell Imaging Experiment
<ol>
	<li>Make sure you have the desired media ready in appropriate syringes connected to flexible plastic tubes with internal diameter of 0.02" (Tygon) in the syringe pump.</li>
	<li>Grow S. cerevisiae strains to the desired phase in liquid YPD. Vortex thoroughly and place 300 &mu;l of each strain into a microcentrifuge tube.</li>
	<li>Gently place 1 &mu;l of Concanavalin A (sigma) 2 mg/ml into each well. Wash out excess Concanavalin A from each well twice by gently pipetting water.</li>
	<li>While the Concanavalin A is drying, wash strains twice with 300 &mu;l of SC medium lacking glucose. Washing residual glucose from the cell walls helps proper adherence to the Concanavalin A.</li>
	<li>Vortex cells thoroughly. Pipette 0.75 &mu;l or less of each cell suspension into its own well (Figure 3). Gently wash out residual cells with rich medium. The next two steps need to be performed quickly in order to prevent cells from drying up.</li>
	<li>Plasma treat the chip and the wells thoroughly using the hand-held corona treater, being careful not to hit the wells directly. Carefully place the chip on the wells, paying close attention to the alignment between the two (this step can be done under a stereoscope). Gently press to adhere the two layers of PDMS. Slowly fill the device completely with about 50 &mu;l rich medium, making sure no air bubbles are left inside the channel.</li>
	<li>Connect the device, inserting the tubes into the appropriate inlets and start media flow. Take care that no air bubbles are introduced into the device as these can be tricky to remove; this can be accomplished by disconnecting an inlet or outlet and gently tapping on the device where the bubbles are, being careful not to break the glass coverslip.</li>
	<li>Place under microscope and find appropriate imaging points in each well.</li>
	<li>Keep the cells under rich medium flow for 1 hr or longer to allow recovery if needed.</li>
	<li>Start the experiment: we use constant flow from each of the two syringe pumps to prevent backflow of the media, changing relative flow rates as desired. Setting pump A to 0.8 ml/hr and pump B to 0.2 ml/hr results in medium A flowing over 80% of the width of the channel (Figure 4). This can be dynamically changed by changing the two flow rates. The new conditions stabilize within seconds along the full length of the channel2.</li>
</ol>

<ol><li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li>
<li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+processing+by+the+HOG+MAP+kinase+pathway%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li>
<li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li>
<li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells%5BTitle%5D)+AND+%22Nat.+Rev.+Genet%22%5BJournal%5D)">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li>
<li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters%5BTitle%5D)+AND+%22Chips+%26+Tips%22%5BJournal%5D)">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips & Tips. , (2010).</li>
<li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PDMS+bonding+by+means+of+a+portable%2C+low-cost+corona+system%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li>
<li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li>
</ol>

No conflicts of interest declared.

In this paper we present a simple benchtop method for creating a microfluidic device that allows following several yeast strains simultaneously under dynamic conditions. Following several strains in one channel provides a reliable tool for comparing dynamics of single cell responses in multiple strains. One advantage of our approach is the ability to fabricate the device with simple techniques without the need for a clean room. In fact, we have also carried out the protocol skipping the plasma treatment operations (in st...

Cells are constantly reacting to a dynamically changing environment, by changing their metabolism, transcriptional profile and cellular functions. To study these phenomena, methods that can quantify such changes are needed. One type of readout for such responses is the change in localization of stress related transcription factors in response to nutritional stress.
Microfluidic devices1 have been used to dynamically manipulate environmental conditions to cells2-4. They present several advantages for live cell imaging: the environment of the cells can be precisely controlled and dynamically changed; cells can be live-imaged at all times, including during manipulation of external conditions; and minimal reagent volumes are required. A very simple design for a microfluidic device, allowing alternation between two conditions, is a Y-shaped device2. We routinely use Y-shaped microfluidic devices that allow application of dynamic changes to the cells in the channel. We use this device to follow localization changes of fluorescently tagged stress related transcription factors in yeast cells. We follow these changes under changing nutritional conditions. For example, we can provide a pulse (or series of pulses) of glucose-containing medium within a period of no-glucose medium, at a very fine temporal resolution. Often in such experiments, one is interested in comparing the responses of different cell strains (for example, different mutants, or different wild isolates). The Y-shaped channel is limited to one strain in each channel - if more than one strain is used, there is no simple way to distinguish between the strains. To overcome this, different channels can be used. If we want to apply the same dynamics conditions to multiple strains, we would like to combine the Y-channel concept with multiple channels. There are two challenges in implementing this solution: It can be hard to apply the same conditions at the same time to all channels, and there is a geometric limitation to the number of Y-channels that can be fitted in one device. We were therefore looking for a way for viewing several strains in one channel under dynamic conditions.
Here we describe a two-layered microfluidic device intended for imaging multiple strains in one channel under the same dynamic conditions. The bottom layer consists of thin PDMS with a row of holes. This layer is attached to the glass coverslip creating wells into which we will place the different strains, adhering them with Concanavalin A to the glass. The second layer is a Y-shaped microfluidic device created using the scotch tape method5. After placing the strains in the wells the second layer is quickly aligned and attached to the first one, creating one channel with several wells that contain the different strains. This final device allows following several strains in one channel, where all strains are subjected to the same conditions at the exact same time. The whole design and production process can be done on the benchtop, using simple means, with no soft lithography.

<table><tbody><tr><td>PDMS- SYLGARD 184</td><td>Dow Corning USA</td><td></td><td></td></tr><tr><td>Vacuum desiccator</td><td>Nalgene</td><td>5310-0250</td><td></td></tr><tr><td>Biopsy punchers</td><td>Ted Pella Inc.</td><td>Harris Uni-Core 15076 (2 mm), 15074 (1.2 mm)</td><td></td></tr><tr><td>Syringe pumps</td><td>Chemyx</td><td>Fusion 200</td><td></td></tr><tr><td>Corona treater</td><td>Electro-technic products</td><td>BD-20</td><td></td></tr><tr><td>Tygon tubing</td><td>Tygon</td><td>S-54-HL</td><td></td></tr><tr><td>Concanavalin-A</td><td>Sigma</td><td>C7275</td><td></td></tr><tr><td>Scotch tape</td><td>3M Scotch</td><td>Transparent Tape 1/2&quot;</td><td></td></tr></tbody></table>

a microfluidic device for studying multiple distinct strains

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

To demonstrate the separation between different strains we imaged two distinguishable yeast strains in alternate wells. Imaging the full wells shows no cell leakage between wells (Figure 5a,b). Both strains have the transcription factor MSN2 tagged with YFP. To test the simultaneous effect of dynamically changing conditions, we switched the flow rates in the two input channels, creating a step of no-glucose medium, which resulted in localization of Msn2-YFP to the nucleus (Figure 5c,d). ...

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.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

The overall goal of the following method is to image the behavior of single cells from different strains of yeast under the same dynamic conditions. This is achieved by fabricating a two layer microfluidic device, or the bottom layer will contain each of the different strains separately, and the top layer will provide the dynamic flow conditions as a second step. Different yeast strains are placed in the wells and the device is closed, creating a single channel with multiple separated strains.Next, we image the cell responses to dynamic medium changes By controlling the flow rates in the two inputs of the Y Channel, the results show different strains subjected to the same dynamic conditions and no cross contamination between wells. This microfluidic device has several key advantages over other existing systems. It allows imaging multiple distinct strains under the same dynamic conditions.Also, importantly, it can be manufactured easily in any lab with no need for special equipment. The Of this method came from discussion on how to follow the responses of different wild East isolates to starvation passes. Start, draw or print out the desired microchannel layout to scale.Next, cover a glass slide with layers of scotch tape To achieve the desired height of the channel, place the layout design on a flat surface and align the slide over the design pattern. Now carefully cut the tape on the glass slide according to the layout. Remove the scotch tape from all regions of the glass slide.Accept those in the layout of the micro channel. Place the slide in a 65 degrees Celsius heating oven for three minutes. Then clean gently with ethanol.Mix the base and curing components of PDMS as recommended by the manufacturer. Pour about 30 milliliters of PDMS mixture into a Petri dish to a height of approximately 0.5 centimeters. Degas the PDMS in vacuum if needed.Submerge the glass slide in the PDMS with the patterned scotch tape facing up. Placing the pattern after the PDMS prevents formation of air bubbles between the slide and dish Bottom cure for 48 hours at 65 degrees Celsius, making sure the dish is horizontal using a bubble level in a new 90 millimeter Petri dish. Pour three milliliters of PDMS mix.This step can be done on a spin coder if available. DGAs and cure as shown earlier. Now gently cut out the flow layer of the microfluidic device to the desired size with a biopsy puncher.Create holes for the inlets and outlets in the flow layer. Also cut out a similarly sized wells layer from the second Petri dish and place on a thick glass. Slide over the design layout.Then punch out wells in alignment with the microchannels on the layout using ethanol, clean both PDMS layers and a glass cover slip and air dry. Next, treat the glass cover slip and the wells layer PDMS with either standard plasma etcher for non reversible bonding or a handheld corona treater for reversible bonding. Carefully place the wells layer on top of the cover slip to cause adhesion.Gently rubbing out any air bubbles. Prepare desired media in appropriate syringes and connect to tigon tubing threaded through a syringe pump. For cell imaging.Use strengths. Go to the desired phase, Vortex the culture thoroughly, and transfer 300 microliters into micro centrifuge tubes. Gently place one microliter of con canna A into each well of the PDMS wells layer.Wash out excess conna A from each well twice by gently pipetting water while the con in A is drying. Wash the strains twice with 300 microliters of SC medium lacking glucose. Washing residual glucose from the cell walls helps proper adherence to the conval.In a after vortexing, the cells thoroughly pipette approximately 0.5 microliters of the cell suspension into individual wells. Gently wash out residual cells with rich medium. The next two steps need to be performed quickly in order to prevent cells from drying up.As an optional step plasma, treat the chip and the wells being careful not to hit the wells directly. Then under a stereoscope, carefully place the chip on the wells paying close attention to the alignment between the two. Gently press to adhere the two layers of PDMS.Slowly fill the device completely with about 50 microliters of rich medium, making sure no air bubbles are left inside the channel. Now connect the device inserting the tubes into the appropriate inlets and start media flow. Take care that no air bubbles form in the tubing as these can get stuck in the device and destruct the flow.Proceed to place the device under a microscope and find appropriate imaging points in each. Well keep the cells under rich, medium flow for one hour or longer if necessary. To allow recovery.Start the experiment with constant flow setting. Pump A to 0.8 milliliters per hour and pump B to 0.2 milliliters per hour. For medium a flowing over 80%of the width of the channel.We can dynamically change the conditions in the device by changing the relative flow rates. The new conditions stabilize within seconds along the entire channel To demonstrate the separation between different strains. Two distinguishable yeast strains are imaged in alternate wells.Note that there is no cell leakage between wells in this experiment. Both strains have the transcription factor MSN two tagged with YFP to test the simultaneous effect of dynamically changing conditions. The flow rates in the two input channels were changed to create a step of no glucose medium.This resulted in localization of MSN two to the nucleus. Importantly, time track of nuclear MSN two YFP levels in single cells can be analyzed. So the PDMS microfluidic device can be used for application of concurrent dynamic conditions to multiple wells.While attempting this procedure, it's important to remember not to let the cells dry out during the assembly of the device. Once master, this technique can be done easily with no need for soft lithography.

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Learn to image single yeast cells from different strains under identical dynamic conditions using a two-layer microfluidic device. This method prevents cross-contamination while enabling simultaneous observation of multiple strains.

A Microfluidic Device for Studying Multiple Distinct Strains

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