Glucose Incorporation into Lipid and Glycogen: Difference between revisions

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filled in details from Brady protocol.
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==Materials==
==Materials==
* Cells plated in 12 well dishes
* Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)
* PBS at 4C
* Borosillicate Test Tubes (VWR)
* 50% KOH - '''Make Fresh'''
* 6 mg/mL Glycogen in 30% KOH - '''Make Fresh'''
* 70% Ethanol, cooled to 4C


==Protocol==
==Protocol==
# Turn heating block up to 100C.  Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
# Starve cells in '''Low Glucose Starvation Media''' for >3h.
# Prepare radioactive glucose solution by combining 1 uCi 14C-Glucose + 50 uL '''Low Glucose Starvation Media'''.  Make at least enough for one extra well.
# Pretreat cells with inhibitors if required.
# Add 100 nM Insulin (or less if needed) and incubate 15 min at 37C in normal incubator.
# After 15min add 50 uL of the radioactive glucose solution, and place in the radioactive tissue culture incubator.
# Save 3 x 5 uL of the radioactive glucose solution to count total radioactivity.
# After 45 min  wash cells 3x with 1 mL of PBS.
# Resuspend cells in 1 mL of PBS.
# Transfer 400 uL of resuspended cells and 5 mL of non-aqueous betaflour scintillant and vortex.  Set aside.
# Transfer 400 uL of resuspended cells to 600 uL of 50% KOH in labelled boroscillicate tubes.
# Add 200 uL of glycogen solution to cells and vortex.
# Heat at 100C for 15 min. 
# Reset cooling block to 85C and incubate an extra 15 min.
# Remove test tubes to a rack.
# Add 2.5 mL of 100% Ethanol and vortex.  Solution should turn cloudy
# Replace in the 85C heating block and incubate for 30 min.
# Place on ice for 15 min to precipitate glycogen.
# Centrifuge 10 min at 3500 RPM.
# Gently aspirate liquid.  If necessary leave a little liquid in the tube.
# Add 3 mL of 70% chilled ethanol.  Recentrifuge for 5 min at 3500 RPM
# Aspirate most of the ethanol and let air dry overnight.
# The next day resuspend the glycogen pellets in 1 mL, and heat at 37C for 30 min to resuspend.  Transfer to aqueous scintillation fluid and count.
# For the lipid, remove the lipid portion to a fresh vial and count.
Adapted from Matt Brady's protocol.

Revision as of 16:07, 24 February 2010

Materials

  • Cells plated in 12 well dishes
  • Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)
  • PBS at 4C
  • Borosillicate Test Tubes (VWR)
  • 50% KOH - Make Fresh
  • 6 mg/mL Glycogen in 30% KOH - Make Fresh
  • 70% Ethanol, cooled to 4C

Protocol

  1. Turn heating block up to 100C. Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
  2. Starve cells in Low Glucose Starvation Media for >3h.
  3. Prepare radioactive glucose solution by combining 1 uCi 14C-Glucose + 50 uL Low Glucose Starvation Media. Make at least enough for one extra well.
  4. Pretreat cells with inhibitors if required.
  5. Add 100 nM Insulin (or less if needed) and incubate 15 min at 37C in normal incubator.
  6. After 15min add 50 uL of the radioactive glucose solution, and place in the radioactive tissue culture incubator.
  7. Save 3 x 5 uL of the radioactive glucose solution to count total radioactivity.
  8. After 45 min wash cells 3x with 1 mL of PBS.
  9. Resuspend cells in 1 mL of PBS.
  10. Transfer 400 uL of resuspended cells and 5 mL of non-aqueous betaflour scintillant and vortex. Set aside.
  11. Transfer 400 uL of resuspended cells to 600 uL of 50% KOH in labelled boroscillicate tubes.
  12. Add 200 uL of glycogen solution to cells and vortex.
  13. Heat at 100C for 15 min.
  14. Reset cooling block to 85C and incubate an extra 15 min.
  15. Remove test tubes to a rack.
  16. Add 2.5 mL of 100% Ethanol and vortex. Solution should turn cloudy
  17. Replace in the 85C heating block and incubate for 30 min.
  18. Place on ice for 15 min to precipitate glycogen.
  19. Centrifuge 10 min at 3500 RPM.
  20. Gently aspirate liquid. If necessary leave a little liquid in the tube.
  21. Add 3 mL of 70% chilled ethanol. Recentrifuge for 5 min at 3500 RPM
  22. Aspirate most of the ethanol and let air dry overnight.
  23. The next day resuspend the glycogen pellets in 1 mL, and heat at 37C for 30 min to resuspend. Transfer to aqueous scintillation fluid and count.
  24. For the lipid, remove the lipid portion to a fresh vial and count.

Adapted from Matt Brady's protocol.

Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php?title=Glucose_Incorporation_into_Lipid_and_Glycogen&oldid=461"