McClean:LFA Membranes

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Overview)
Current revision (23:17, 1 July 2013) (view source)
(Membrane and FCS2)
 
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 thin oval membrane on a 40 mm coverslip.
+
#Position a .75mm-thick gasket on a 40 mm coverslip.
-
#Add 20 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.  
+
#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.  
#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).
#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 THICK gasket and cells already loaded with ConA.  This takes a bit of finesse!  
+
#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.
#Assemble FCS2 as per standard instructions, making sure to keep all pieces dry.

Current revision

Contents

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

  1. Heat 1mL LFA aliquots for ~5 min at 100°C until liquid.
  2. Position a .75mm-thick gasket on a 40 mm coverslip.
  3. 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.
  4. Once dried (~5min) remove one of the coverslips and the gasket.
  5. Remove any excess agar from the sides of the glass slide until a nice circle is left (usually using a pipette tip).
  6. 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!
  7. 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!

  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.

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

or instead, discuss this protocol.

Personal tools