I am trying to set up the glucose uptake assay on 3t3-l1 adipocytes. I have tried insulin conc. from 1nM-1uM for 10min, 30min and 2hrs. I just couldn't see any insulin-dependent stimulation. I am using the following kit for glucose uptake assay:
http://www.funakoshi.co.jp/data/datasheet/CAY/600470.pdf
2-NBDG isn't particularly insulin responsive, we used it as a positive control in L6 for a study using oestrogen analogues and it caused a small increase in fluorescence.
3T3-L1 cells have a lot of GLUT1 in addition to GLUT4, thus basal glucose uptake is high. You won't see 10-fold stimulation like you could in isolated adipocytes. In my hands 2-fold is about what you can get with differentiated 3T3-l1s. The concentration of your glucose analog cannot be too low because of high basal glucose uptake. Do you have a control to make sure your insulin is working properly? Sometimes it is easier to just stop by your local Walgreens and buy a 10 mL bottle of U-100 regular human insulin (no prescription needed; where I am it runs about $90). You can convert U/mL to nM fairly easily.
It's the same western procedure as we have published in J Lipid Res in 2012. Thompson et al. DOI: 10.1194/jlr.D031286
Phosphorylation of T308 is more sensitive in my hands than S438 even signal is lower. I got my phospho- and total Akt antibodies from Cell Signaling.
Dear JP Yang, Can you share with me the protocol for insulin stimulated glucose uptake using 2 NBDG in 3T3 L1 cells..as I am too suffering with no difference in signal when using insulin to stimulate the cells.
Here is what i have tried,
> Differentied cells received serum starvation on DMEM low glucose glutaminate, sodium pyrovate with 0.5% BSA for 2hrs
> wash cells in DMEM glucose free DMEM medium with sodium pyrovate and glutmate +0.5% BSA once.
> incubate cells + insulin in above DMEM glucose free medium containing 100ug/ml 2-NBDG for 30min
> centrifuge the plate for 5min at 400g at room temperature
> aspirate supernatnat
> Add 100ul of cell-based assay buffer to each well
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