McClean: FCS2 for Cycling

Overview

This is the best (so far) protocol we have found for inducing metabolic cycling in the FCS2 chamber. The low-fluorescence agar pad is used to keep the cells trapped against the coverslip during growth to allow long-term observation.

Materials

Low Fluorescence Media (Recipe) of three different types:

• Full-nutrient (for growing up your culture)
• Starvation media (we used LFM with glucose removed)
• Cycling media (we used LFM with 0.001% glucose)

FCS2 chamber system, including:

• Peristaltic pump
• Three sections of tubing
• Three coverslips
• Metal chamber base
• Plastic chamber top
• Microaqueduct slide
• Two .75mm-thick oval gaskets
• Circular top gasket
• Media and effluent containers

Additional materials:

• 1mL aliquot Low-fluorescence agar
• Aliquot of concanavalin A
• Syringes (16G1 needles)

Stock Solutions

Low-Fluorescence Media Use the LFM Recipe as a base, removing nutrients you wish to limit.

Low-Fluorescence Agar Similarly, we used the glucose-free version of the LFA, of which Anjali made numerous aliquots.

Concanavalin A We use the standard ConA (protocol), (not the ion-free).

Protocol

Culturing the cells

1. Set back overnight culture and allow to grow back to about OD 0.5 in your growth LFM.
2. When cells have reached appropriate OD, spin down and re-suspend in the starvation LFM. Incubate for at least 4 hours.

Chamber set-up

1. Heat LFA at ~95°C.
2. When liquified, apply 40μL to center of first coverslip, place oval gasket around agar, and place second coverslip on-top, pressing slightly to spread the agar.
3. Apply ~20μL of ConA to the third coverslip and let incubate for ~5 minutes.
4. Aspirate off the ConA and add 20μL of diluted starved cells. Incubate for ~5 minutes.
5. By this time, the LFA should be solidified -- remove the top coverslip and gasket, and carefully slide pad to edge of glass plate to be ready to move it.
6. When cells are ready, rinse using a syringe and ~3mL of the starvation media to dislodge any non-stuck cells. Then aspirate.
7. Slide the agar pad onto the adhered cells.
8. Place the coverslip, now with cells and pad, in the chamber, which should be already loaded with media.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

--Stephanie S. Steltzer 16:15, 22 July 2015 (EDT) Using Bioptechs software rundown: Be sure to have pumps set at 10 and 100 and switch to EXT. After the system is completely set up, open Windows XP Mode under Windows Virtual PC to run the emulator. Open the application Bioptechs PTC. At the top of the screen under devices, be sure to attach the unidentifiable device that is the USB connected interface box. Do not forget to calibrate the pumps (see calibration manual). tip: If using the software for the first time on a new computer, download Windows Virtual PC and Windows XP Mode. Make sure the computer you are using has the driver DT9812; if not, you may need to update the drivers on the computer. Follow instructions in manual from there, installing the software and data translation device from the disk.

--Bennett A. McIntosh 14:27, 5 August 2013 (EDT) I found it useful to keep the incubating ConA/cells somewhere other than out in the open -- an old pipette tip box, for example.

--Bennett A. McIntosh 14:27, 5 August 2013 (EDT) It is often easier to move the agar pad after rinsing carefully with DI water.

--Bennett A. McIntosh 14:44, 5 August 2013 (EDT) I load the media into the chamber system by pushing it in with a 3mL syringe and a 16G1 tip, since this works much more quickly than waiting for the pump to work.

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

B.P. Tu et al. (2005) Logic of the Yeast Metabolic Ccycle: Temporal Compartmentalization of Cellular Processes. Science 310:1152=1158.

Contact

• --Bennett A. McIntosh 14:27, 5 August 2013 (EDT)

or instead, discuss this protocol.