McClean:LFA Membranes

Overview

For use with FCS2 from Bioptechs to keep cells S. cerevisiae monolayer during long experiments. Adapted from Wong et al (2010). We chose to use LFM Agar to minimize florescence from the membrane and removed carbon sources for the purpose of our experiments. Media can be poured in any type of mold.

Final Composition of Media (100 mL total)

Chemical

10 ml of 10X nitrogen source (ammonium sulfate or alternative)

10 ml of 10X potassium phosphate

10ml of 10x amino acid supplement

1ml of 100X MgSO4

.5ml of 200x NaCl

.5ml of 200x Ca2Cl

100μL of 1000X metals

50 μL of 2000x vitamins

17.8 ml of sterile H20

50 ml 2X bacto agar

Note: 2X bacto agar: 20g of bacto agar brought up to 500ml of liquid, autoclaved, microwave to melt for use. For all other stock solutions see LFM Recipe.

Membrane and FCS2

1. Heat 1mL LFA aliquots for ~5 min at 100°C until liquid.
2. Position a thin oval membrane on a 40 mm coverslip.
3. Add 160 uL of the liquid media directly onto the cover slip, spreading with pipette as you go, and cover the top with another 40 mm coverslip. Apply slight pressure to cause the membrane to spread out. (With a 22mm x 14mm x 0.5mm gasket, the volume will be about 150 uL -- you can work it with this much, but a bit of extra volume allows for some leakage)
4. Once dried (60 mins? Overnight?) remove one of the coverslips and the gasket.
5. Remove any excess agar from the sides of the glass slide until a nice oval is left (usually using a pipette tip).
6. Slide the membrane off of the original coverslip onto a coverslip that has the THICK gasket and cells already loaded with ConA. This takes a bit of finesse! Here it is often helpful to flush the membrane with a bit of water, then dry, before carefully pushing the agar off of one coverslip and onto the other.
7. Assemble FCS2 as per standard instructions, except apply the oval gasket/membrane/coverslip as one piece.

Notes

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

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

Wong, I et al (2010) An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study Lab Chip 10 2710-2719

Contact

• Anjali Bisaria 14:01, 20 July 2011 (EDT)

Or *Bennett A. McIntosh 11:08, 21 June 2013 (EDT):

or instead, discuss this protocol.