McClean:LFA Membranes: Difference between revisions
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==Overview== | ==Overview== | ||
For use with FCS2 from Bioptechs to keep | For use with FCS2 from Bioptechs to keep ''S. cerevisiae'' cells 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)== | ==Final Composition of Media (100 mL total)== | ||
| Line 35: | Line 35: | ||
==Membrane and FCS2== | ==Membrane and FCS2== | ||
#Heat 1mL LFA aliquots for ~5 min at 100°C until liquid. | #Heat 1mL LFA aliquots for ~5 min at 100°C until liquid. | ||
#Position a | #Position a .75mm-thick gasket on a 40 mm coverslip. | ||
#Add | #Add 40 uL of the liquid media directly onto the cover slip, and cover the top with another 40 mm coverslip. Apply slight pressure to cause the membrane to spread out. | ||
#Once dried ( | #Once dried (~5min) remove one of the coverslips and the gasket. | ||
#Remove any excess agar from the sides of the glass slide until a nice | #Remove any excess agar from the sides of the glass slide until a nice circle is left (usually using a pipette tip). | ||
#Slide the membrane off of the original coverslip onto a coverslip that has the | #Slide the membrane off of the original coverslip onto a coverslip that has the .75 mm gasket and cells already loaded with ConA, and rinsed to remove any unadhered cells. This takes a bit of finesse! | ||
#Assemble FCS2 as per standard instructions, | #Assemble FCS2 as per standard instructions, making sure to keep all pieces dry. | ||
==Notes== | ==Notes== | ||
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | <!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | ||
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | ||
#List troubleshooting tips here. | #List troubleshooting tips here. | ||
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | #You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | ||
Latest revision as of 20:17, 1 July 2013
Overview
For use with FCS2 from Bioptechs to keep S. cerevisiae cells 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
- Heat 1mL LFA aliquots for ~5 min at 100°C until liquid.
- Position a .75mm-thick gasket on a 40 mm coverslip.
- Add 40 uL of the liquid media directly onto the cover slip, and cover the top with another 40 mm coverslip. Apply slight pressure to cause the membrane to spread out.
- Once dried (~5min) remove one of the coverslips and the gasket.
- Remove any excess agar from the sides of the glass slide until a nice circle is left (usually using a pipette tip).
- Slide the membrane off of the original coverslip onto a coverslip that has the .75 mm gasket and cells already loaded with ConA, and rinsed to remove any unadhered cells. This takes a bit of finesse!
- Assemble FCS2 as per standard instructions, making sure to keep all pieces dry.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
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)
- Bennett A. McIntosh 11:08, 21 June 2013 (EDT):
or instead, discuss this protocol.
