McClean:LFA Membranes: Difference between revisions
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(New page: ==Overview== Use this protocol to make small Low Fluorescence Agar Membranes. Adapted from Wong et al (2010) for use with the FCS2. We adapted the original protocol for bacterial membran...) |
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==Overview== | ==Overview== | ||
Use this protocol to make small Low Fluorescence Agar Membranes. Adapted from Wong et al (2010) for use with the FCS2. We adapted the original protocol for bacterial membranes for different carbon sources and for ''S. cerevisiae''. | Use this protocol to make small Low Fluorescence Agar Membranes. Adapted from Wong et al (2010) for use with the FCS2. We adapted the original protocol for bacterial membranes for different carbon sources and for ''S. cerevisiae''. | ||
==Overview== | |||
For use with FCS2 from Biopteks to keep 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)'''== | ||
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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 [McClean:LFM_Recipe]. | 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 [[McClean:LFM_Recipe | LFM Recipe]]. | ||
==Membrane and FCS2== | |||
#Heat 1mL LFA aliquots for >3 min at 100°C until liquid. | |||
#Position a thin oval membrane on a 40 mm coverslip. | |||
#Add 100 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 remove one of the coverslips and the gasket. | |||
#Remove any excess agar from the sides of the glass slide until a nice oval is left (usually using a pipette tip). | |||
#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! | |||
#Assemble FCS2 as normal. | |||
Revision as of 10:49, 27 July 2011
Overview
Use this protocol to make small Low Fluorescence Agar Membranes. Adapted from Wong et al (2010) for use with the FCS2. We adapted the original protocol for bacterial membranes for different carbon sources and for S. cerevisiae.
Overview
For use with FCS2 from Biopteks to keep 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 >3 min at 100°C until liquid.
- Position a thin oval membrane on a 40 mm coverslip.
- Add 100 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 remove one of the coverslips and the gasket.
- Remove any excess agar from the sides of the glass slide until a nice oval is left (usually using a pipette tip).
- 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!
- Assemble FCS2 as normal.
