Mouse adipocyte glucose uptake assay - Protocol for glucose uptake assay in mouse adipocytes

Procedure

Day 0 cell culture:

1. Grow 3T3-L1 cells in high glucose DMEM + 10% Calf Serum + P/S/F in 6-well plates.
2. After cell have reached complete confluent, replace growth media with stimulation media containing:

High glucose DMEM:
10% FCS, P/S/F
1 µl / ml of Insulin (10 mg/ml, 1000x in HEPES, final 10 µg/ml)
1 µl / ml IBMX (3-isobutyl-1-methylxanthine, 500 mM stock, 1000x), final 0.5 mM
0.1 µl / ml Dexamethasone (10000X in ethanol, final 1 µM).
Day 3

1. Replace with fresh media containing Insulin only, no IBMX, no Dexamethasone.  Change the media on day 6.  Cells will be differentiated on day 7-9.

Day 6
Cells will be cultured in serum-free DMEM for 2 hr ± treatments

1. Incubate cells  in 1 ml of KRH with 100 nM insulin for 20 min ± treatments.
2. Add 1 µCi of 2-deoxy-D-[2,6-3H]-glucose (50 µl in volume) to each well for 10 min.
3. Wash cells with KRH buffer 3X.
4. Lyse cells in 1 ml of RIPA buffer. 
5. Count 300 µl.

KRH buffer:
25 mM HEPES-NaOH [pH 7.4]
120 mM NaCl
5 mM KCl
1.2 mM MgSO₄1.3 mM CaCl2
1.3 mM KH₂PO₄
References
J Clin Invest.  2005, 115, 291-301.

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Dr. Guo-Ping Shi
Harvard Medical School
Brigham and Women's Hospital
Cardiovascular Medicine
Boston, MA