Difference between revisions of "Glucose Uptake Assay"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m (added categories) |
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#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3 | #Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3 | ||
#Do a bradford assay on 20 uL of cells (use PBS as blank) | #Do a bradford assay on 20 uL of cells (use PBS as blank) | ||
| + | |||
| + | ==Analysis== | ||
| + | *For excel fill in orange cells in this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/Excel%20Templates/2-DG%20Template.xlsx] | ||
| + | *For sweave use this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/R/Sweave/2-dg%20analysis.Rnw] | ||
[[Category: Metabolic Measurements]] | [[Category: Metabolic Measurements]] | ||
[[Category: Glucose Uptake]] | [[Category: Glucose Uptake]] | ||
[[Category: Cell Culture]] | [[Category: Cell Culture]] | ||
Revision as of 13:44, 3 November 2011
Materials
- 2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in KRBH Buffer
- 0.5% BSA in KRBH Buffer
- Radioactive 14C-2-deoxyglucose
- Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG
Protocol
- Starve cells >3h in 0.5% FBS
- Prepare insulin in KRBH/BSA (0.6uL insulin/mL, needing 0.5 mL per well)
- Wash cells 2x with warm PBS -/-
- Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements
- Wait 30 min
- Add 50 uL hot 2-DG solution per well and start timer
- After 5 min add 50 uL cold 2-DG
- Wash cells 3x1mL with cold PBS
- Add 500 uL PBS per well
- Scrape cells with an upside down p200 tip
- Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
- Do a bradford assay on 20 uL of cells (use PBS as blank)
