I’ve followed different recent papers to measure glucose uptake in 3T3-L1 cells and L6 cells. The problems I found are (1) that fluorescent values obtained are small and (2) it’s hard to see the differences between insulin (100nm)-treated and insulin non-treated cells. Does anyone have an exact protocol I could follow where these problems don't occur?
We have published some articles regarding the 2-NBDG uptake in the related cell line 3T3-F442A. I am sending you the manuscript where the protocol is described. I hope it can be useful
I had the same problem using C2C12 myotubes but the signal is alos low.
It should be better with adipocytes: maybe you can try to increase the starvation without serum before exposure to 2NBDG (3h or more?) : did you use a low glucose DMEM for your experiment? I'm interested by your experiment since I need to perform the measurement on my 3T3 also or primary adipocytes from rat! [email protected]
I would say that time is important. Are you on a confocal microscope or is this a single time point on a plate reader?
Our protocol worked well on neurons.
http://www.sciencedirect.com/science/article/pii/S0143416011001746
Thanks. Dear Olivier, I read your paper. I will try to measure glucose uptake at different time intervals. I measured glucose uptake at a single time point on a plate reader. Any suggestions?
Dear Angel Alonso-Castro, I thank you for your paper. A few months ago, after I saw your paper, I tried to establish that protocol in my lab, but the problem I have is no signal difference on insulin treated and non-treated cells. I have followed the same protocol like yours. Before measuring fluorometry, I carefully washed the 96 black-well plate with PBS, to remove extracellular 2-NBDG. If you share your protocol in detail, I would be really grateful to you. Please, my email: [email protected]
Dear Frederic, I tried using low glucose, no glucose, and serum free medium, but I couldn't find much difference (that many papers are reporting) on signaling.
In my experience, 3T3 is far better than skeletal muscle cells (L6) for glucose uptake measurement.
I have been trying different conditions like you did. my findings are basically the same as you, 1) low signals, 2) no detectable difference. i also tried 3t3-l1 -F422A. the same story.
However, i was able to detect great insulin simulated glucose uptake on 3t3-l1 cells by measuring the decrease of extracellular glucose level during insulin stimulation. I started to see the difference after 6hrs.
what is the typical starvation time for cells before you add 2-NBDG?
Dear Prince, I starved my cells for 3h before adding insulin.
Best regards,
Frederic
Hello, I am interested in measuring glucose uptake in C2C12 myotubes using [3H]2-deoxyglucose. Although I am not using 2-NBDG, I too am finding no detectable difference between insulin-stimulated and non-stimulated conditions. I have tried different insulin concentrations (10nM, 100 nM, 1 µM), but still see no insulin effect Does anyone have an exact protocol using [3H]2-deoxyglucose where they consistently have an insulin effect?
There is very little GLUT4 in C2C12 myotubes so it is normal that your insulin response will be low. Try to serum-starve your cells overnight. For C2C12 and L6 we routinely do the following serum starvation:12 hours serum starvation in DMEM or alfa MEM + 0.1% BSA overnight in CO2 incubator, next day wash cells with 0.1 ml /well with DMEM (no bicarbonate) or alfa MEM/ 20mM Hepes / 0.2% BSA or use Krebs Ringer Phosphate Buffer supplemented with 0.2% BSA and incubate the cells for 1 hour at 37degC. Then do the insulin stimulation or drug treatment - typical insulin stimulation 200 nM insulin for 30 min.
Dear JP Yang, Can you please share your protocol for insulin stimulated glucose uptake using 2 NBDG in 3T3 L1 cells. My email ID: [email protected]
I am interested in measuring glucose uptake using L6 cells. For how long do I serum-starve them?
Does anybody know why we need two steps of starvation in glucose uptake assay. First with serum-free cell media and then with KRP. What is the purpose of starving with KRP after DMEM.
I've fould a BSc thesis, which do answers some questions asked here. The thesis: http://scholarworks.uark.edu/cgi/viewcontent.cgi?article=1034&context=bmeguht
Need a protocol for insulin stimulated glucose uptake using 2 NBDG on HepG2 cells.
Hi Achille,
We have recently develop a few glucose uptake assays which you might try it out. one of them is NBDG based (see the 2nd link). You might try the insulin stimulation protocol based on the 3rd link. Thanks! and good luck!
https://www.aatbio.com/search?narrow=0&query=glucose+uptake
https://www.aatbio.com/products/cell-meter-2-nbdg-glucose-uptake-assay-kit?unit=23500
https://www.aatbio.com/products/protocol/36500.pdf
Best regards,
Jennifer
Thanks Jennifer. i think now i have an idea on how to perform this assay
Hi guys! I am trying to measure glucose uptake in AC16 cells (a commercial cardiomyocytes cell line), but I did not see an increase in glucose uptake upon 100nM insulin stimulation! wondering if you guys have any tips to perform this assay? thank you.
i did the experiment in this way:
serum-free treatment for 48hours, and followed by serum/glucose-free medium incubation for 1 hr, and then 100nM insulin/450uM 2NBDG for 1hr.
Hi Eugene,
Please try our cat#23500 see how it goes. Thanks!
https://www.aatbio.com/products/cell-meter-2-nbdg-glucose-uptake-assay-kit?unit=23500
Best regards,
Jennifer
Pratik Ramjivan Shriwas KRP is used to starve the cells of glucose. Since we want to measure the exogeneously supplied 2NBDG.
Hi, Does anyone have experience on B16 or 4T1 cell lines with 2NBDG and in the staining process the free glucose medium does always have to have BSA? and this medium could be PBS?
Bhesh Raj Sharma Have you found a working protocol for insulin stimulated glucose uptake in 3T3-L1 cells? I am having similar problems and no idea what I'm doing wrong.
Hi! I would recommend doing a dose-dependent assay. Start from as low at 5 nM and go up to 100 nM of insulin treatment. Sometimes, excess insulin results in insulin resistance and you end up seeing no or very little uptake.
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