I have been working with the C2C12 myotubes and measured their glucose uptake activity using an indirect glucose uptake assay (measuring how much glucose is left in the medium after stimulating the cells with stimulators). I am trying to switch to using 2-NBDG. However, the experiment did not seem to work. We could not get a high-level of NBDG uptake, although we have no problem with cell lysate. Here is how we did:

1. Differentiated the cells for 4 days using 2% HS

2. With the differentiated cells, washed the cells once with 1xPBS and replenished the medium with a serum-free DMEM medium containing 1% BSA for 18 hours

3. Washed the cells 3 times with 1xPBS and incubated the cells with Krebs Ringer phosphate HEPES (KRPH: 20 mM HEPES, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, pH to 7.4) with 100 nM insulin (final concentration) or metformin for 30 minutes

3. Replaced the medium with 2-NBDG 100 uM into the well and further incubated the plate at 37°C with 5% CO2 for another 30 minutes

4. Lysed cells with T150 buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM Benzamidine, 0.1% NP-40).

5. Performed fluorescence measurement (485/550) and Bradford assay for protein

I wonder if:

1. I should not use the KRPH buffer?

2. the amount of insulin or NBDG is not optimized?

Please help if you have an experience or expertise on this experiment.

Thank you very much.