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.
