I got a trouble recent days that the glucose uptake with 170 nM insulin was always no more than 3 times as that in blank control, while my professor demended at lest 6 times. I've repeated in different conditions as far as I learned from lots of articles. However, no progess has been got.
So will some one share me any useful experience? Besides, my 3T3-L1 always float after differentiation for 8-9 days, does it means any mistake as some articles said that mature 3T3-L1 adipocytes can be maintained to day 12-15 after differentiation?
2/16 added: The glucose uptake assay is KRP-hepes: 10 mM HEPES, 137 mM NaCl, 4.7 mM KCl, 1.22 mM MgSO4, 2.5 mM CaCl2 and 2.5 mM KH2PO4. Assay will be performed before glucose starvation for 30 min.
Perhaps you could try to reduce the concentrations of the other substrates that might be present in the cell media such as Glutamine, Fatty Acids, and Pyruvate. Eventually, cells will try to maintain a steady production of ATP and might start to use more Glucose (this might work unless there are limits or problems with glucose transport or any other components of Glucose metabolism machinery). Good luck!
How long you keep the cells in KRP-Hepes before you add glucose (and how much you add 25mM) for the uptake measurement?
How much glucose do you add for the assay? What grade (purity) are the chemicals used for the prepping the KRP-HEPES and cell media. In the past, I was pH-ing a media for Seahorse XF experiment with NaOH that was made in the lab from solid NaOH with not so great purity and it was reducing dramatically basal OCR of the cells. After a couple of unsuccessful assays, we switched to cell culture tested high purity stock solution of NaOH from Sigma and all these problems disappeared.
Oh, your questions made me realized the deficiency in my description. ╯﹏╰
Well, all chemicals are from sigma. The protocol is as blow:
3T3-L1 adipocytes were grown to confluence and differentiation for 8 days in 24-well multiplates. After serum starvation for 4 hours, cells were incubated in KRB-HEPES buffer (10 mM HEPES, 137 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2 and 2.5 mM NaH2PO4, all sigma) with indicated concentrations of BSA (pH 7.4) for 30 min. The cells were stimulated with various concentrations of human insulin (0–170 nM, Sigma) for 15 min, and then 2-[3H]DOG (1 uCi/mL, nearly 5 uM) plus 2-DG (i've tried 0.05, 0.1, 0.5, 1 mM) was added for 10 min. The reaction was stopped by the addition of 10 mMcytochalasin B. The cells were washed three times with ice-cold PBS and solubilised with 0.2% SDS–0.2M NaOH. The radioactivity incorporated into the cells was measured by liquid scintilla- tion counting (Perkinelmer). The results were corrected for non-specific uptake determined by 2-[3H]DOG uptake in the presence of 10 mM cytochalasin B. (Mostly copy from doi:10.1016/j.diabres.2008.05.007) And the non-specific uptake was always nearly 20%-30% of that in the presence of 170 nM insulin.
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