Assignment 10, Part 1: Measuring the osmotic shock response of yeast

Assemble a microfluidic device

Follow the instructions in Assignment 8 to assemble a PDMS/tape device. Note that you should still have your slab of cured PDMS left over from Assignment 8, so you don't have to repeat that step.

We'll reuse the tubing you cut in Assignment 8, with some slight modifications. We want to be able to wash the flow channel with ethanol, water and ConA solutions before hooking it up to the media reservoirs, so we'll connect some short sections of tubing to the device before hooking it up to the media reservoirs.

1. For each inlet tubing saved from Assignment 8, cut the shortest section of tygon tubing roughly in half.
2. Connect a second ~1 cm length of PEEK tubing to newly cut short sections of tygon tubing, then insert these in the inlet holes of the PDMS.
3. Similarly, cut the outlet tubing to be roughly 1.5 - 2" long, and insert the orange PEEK tubing into the outlet hole.

Incubate device with ConA

We need to immobilize yeast cells to the coverslip of the flow device in order to image them over long times. A standard method of immobilizing yeast cells for microscopy is to coat the coverslip with Concanavalin A (ConA) - a lectin that binds to cell surface saccharides.

1. Thaw 1 aliquot of ConA (250 uL of 1 mg/ml in PBS), preferably slowly, on ice.
2. Use a 1 ml syringe to wash the device with about 500 uL of 70% ethanol (push the solutions backwards through the device, from the outlet to the inlet), then with ~500 uL DI water.
3. Load the ConA solution into a new 1 ml syringe. Without introducing any bubbles, load the entire aliquot of ConA through the device. Leave the syringe connected an let incubate for at least 20 minutes.

Set up media reservoirs and prime inlet lines

1. Insert the high and low salt media reservoirs into the 50ml conical tube holders.
2. Feed the long length of inlet tubing (tygon-silicone-tygon) into the high-salt reservoir. Make sure the end of the tube is at the bottom of the reservoir. consider taping the tubing down to prevent it from getting dislodged.
3. Connect a 1 ml syringe to opposite end of the tubing using a 23G blunt-tipped needle, and pull the fluid from the reservoir into the tubing. Leave the syringe connected.
4. Engage the solenoid valve (i.e. OpenHighSalt), then feed the flexible silicone section of tubing into the now-open slot of the pinch valve.
5. Close the pinch valve to prevent flow from the high-salt reservoir, then disconnect the needle and syringe.
6. Repeat step 4 with the low-salt reservoir. Feed the tubing into the normally-open slot of the pinch valve. Leave the syringe connected until ready to connect the PDMS device.

Connect tubing to device and load yeast cells

The final setup steps include connecting the fluidic device to the reservoirs, and loading the yeas cells into the device. Care should be taken to avoid introducing bubbles, or applying too much pressure once the yeast cells are immobilized.

Prepare yeast cells

1. Check OD of yeast culture - it should be 0.4-0.6
2. Add 2 ml of culture to a 15 mL conical flask, and add 2 ml of YPD.
3. Spin on max for 3 minutes. (or in microcentrifuge on 3000g for 2 min??)
4. Aspirate supernatant and resuspend pellet in 125 uL of SC medium. Transfer culture to a microcentrifuge tube.

Connect device to fluid reservoirs

1. Attach the high salt reservoir tubing to one inlet tube of the PDMS device.
2. Engage the pinch valve to open the high salt valve and close the low salt valve.
3. Disconnect the syringe from the low salt tubing and connect it to the remaining inlet tubing of the device.
4. Connect the syringe to the outlet of the device, and pull some fluid from the low salt reservoir through the device.
5. Engage the valve to open the high salt reservoir and pull some of the high salt medium through the device.
6. Toggle the valves several times and try to remove any visible bubbles by pulling on the syringe.

Load the yeast cells into fluidic device

1. When you’re confident that the tubing and device are bubble free, load the yeast cells into a separate syringe.
2. Ensure the pinch valve is disengaged (low salt) and slowly load the yeast cells into the device, trying not to introduce any bubbles.
3. Leave the syringe connected, and let the cells settle and adhere to the coverslip for 10-15 minutes
4. When ready, disconnect the syringe and connect a long section of tygon tubing to the outlet and a waste reservoir.
5. You should see excess yeast cells flowing through your device once the outlet is connected. You may need to raise the inlet reservoirs to initiate flow.
6. Toggle the valve several times to ensure that both media reservoirs are flowing as expected.