Hi. I have used 2-NBDG to study glucose uptake in C2C12 cells effectively. I starved the cells in no glucose, without FBS media for 2 hours followed by the incubation of the cells with or without insulin for 15mins. I then incubated the cells with 75uM 2-NBDG for 30 mins in no glucose media. I got better stimulation when I co-incubated the cells with NBDG and insulin. Hope this helps.

I'm facing the same problem as you Lyle, just on human primary myocytes, did you managed to solve the problem so far?

Yes I have... it turned out that my cells were bad (from ATCC) and the equipment I was using was bad. I ordered cells from a different company (Sigma) and am using a new plate reader. So I guess it never was anything about the procedure I was doing wrong.

4 days normal differentiation media (high glucose DMEM, HEPES, Glutamax, 2%HS), then 24h in low glucose diff media (same as before just low glucose), then overnight in low glucose, serum free in 1%BSA. The day of the experiment its simply 30 min in 400uM 2NBDG, with or with out insulin, in NO glucose DMEM. Afterwards, 3 washes in COLD PBS+ (with Calcium), then either lyse and read in a plate reader, or fix, mount and image. The low glucose exposure is important because the concentration of glucose in the high glucose DMEM makes them insulin resistant.

One thing that I found interesting is that insulin stimulation seems to be biphasic... meaning a little stimulation will give an effect, a lot of stimulation will reverse it. I'm finding max stimulation at 5-20nM of insulin, and a decrease in stimulation at 100nM.. So make sure your dose-response curve includes those ranges.

Also worth noting that the inclusion of pyruvate in differentiation media seems to weaken the response, and its worth seeing if the inclusion of BSA in the media with 2NBDG and insulin enhances the effect. Good luck!

Hi Lyle,

As I am facing the same problem with C2C12, your answer is really helpful. thanks! I will try your methods definitely!

Oh, I've got one question Lyle. The bad cell line from ATCC, what passage did you use mostly? C2C12 properties change over passages, and I avoid using the cells older than 11 passages.

I couldn't get any passage to consistently differentiate with the cells from ATTC. Every passage from the cells I got from Sigma differentiated every time. I used passages 3-8 for the sake of consistency in my experiments.

Thank you, Lyle!!

I have a problem with the glucose uptake 2-NBDG in C2C12 myotubes. I starved my cells 2 hours without glucose. After that, I treated them with 100nM and 20nM insulin for 15 min then I added my 2-NBDG. My question is, do you have lysed your cells after the test? Because in the kit that I am using, they advise to wash and read the absorbance without lysing the cells but I don't have results.

I tested the assay with and without lysing the cells, and while I still got a clear result with out lysing, the using of a lysis buffer significantly improved the assay.

Also, I would experiment with what you do before you get to the assay. The protocol I listed above is a few years old, one of the first protocols I tried, and have since modified it a lot. I found it was very import to subject the cells to low-glucose DMEM for at least a day, and serum starvation overnight prior to the assay (serum starvation for at least 4 hours, but overnight also worked). I found that glucose starvation didn't do much, but it was critically important to wash all glucose away prior to the assay. I also did 30min incubation with both insulin AND 2NBDG in glucose- and serum- free DMEM. Also, my results showed that 100nM of insulin is actually too much. My highest response was at 25nM of insulin, with less of a response at 50nM and even less at 100nM.

Thank you for your feedback.

It's the first time that I used the glucose uptake 2-NBDG kit from Cayman. I followed their protocol without any incubation in glucose free or serum free medium. I tried the incubation with glucose free medium 2 hours but the medium contain 1% HS and I think that it is not sufficient for the cells. I will try to starve my cells OV with low glucose medium and serum starvation. I hope that it will work with me.

Thank you again!!

One thing to keep in mind is that HS and FBS have insulin in it, so without a serum starvation step your control samples will still be affected by insulin to some degree. In fact, 10% FBS in the media without added insulin has a greater glucose uptake effect compared to insulin by itself.

I also forgot to mention, during my overnight serum starvation step I did add 1% BSA.

I followed your protocol but I don't have any difference in my absorbance between my non stimulated well and insuline 20nM for 30 minutes. I would like to know what kit did you use? What is the reference of 2-NBDG that you used.

I didn't use a kit. This work is from my dissertation which is not published yet, so I attached an abbreviated version of my materials and methods section for your reference. The 2NBDG I used is from ThermoFisher (link below).

https://www.thermofisher.com/order/catalog/product/N13195

Make sure you are measuring FLUORESCENCE and not ABSORBANCE!

Thank you very much for your help and for your materiel and methods. I measured absorbance and not fluorescence. I will try again

THANK YOU!!!

I am struggling since last 8months regarding uptake of 2-NBDG in A549 cell line. As my cells are not uptaking 2-NBDG. I repeated this experiment so many times but I always got the fluorescence like 0.1, 0.09, 0.2 .

The protocol which I am following is-

1. Seed A549 cells in 24-well plate till it become 80-90% confluent.

2. Wash the cells with pbs once and with 0.5% serum DMEM.

3. Incubate the cells with 0.5% serum DMEM for 16-18 hours.

4. Wash the cells with Ringer buffer ( serum and glucose free) twice and then incubate with ringer buffer for 1hour.

5. Incubate the cells with 200 micromolar 2-NBDG in RINGER buffer for 30minutes.

6. Wash the cells with chilled PBS twice and then add RIPA lysis buffer.

7. Keep the plate on ice for 20minutes.

8. Freeze - thaw the cells twice in -80 and 4 degree.

9. scrape the cells and then spin @ 12,000g for 5min at 4 degree

10.transfer the supernatant in black 96-well plate and then take the fluorescence @ 485 and 535 nm in plate reader

I am attaching the reading of my experiment

Those values in the image you attached actually look normal for a 2NBDG glucose uptake assay (except for those two at the bottom right corner, but that happens sometimes). Your protocol looks good, but if you are not getting any differences between experimental groups it may be how you are setting up the plate and how you are normalizing your data.

First off... Do you have any negative controls on your plate? There is always some autofluorescence from the cells, the buffer, and the plate, so its important to subtract that out from the rest of your values. Whenever I set up an experiment, the bottom row of wells never received any 2NBDG, yet still displayed fluorescence values, which were averaged and then subtracted from the rest of the values. For example, a typical reading I would get from a blank well (cells and buffer but no 2NBDG) would be around 0.3, and a value from a sample with 2NBDG would be about 0.4, or up to 0.45 for insulin stimulated. Subtract the autofluorescent values from the experimental values and the differences between your samples will become more evident. (0.4 to 0.45 is nothing, but 0.1 to 0.15 is a 50% increase).

Second, you have to do a total protein assay on those sample samples to control for differences in growth between wells, and divide the raw fluorescence values by the total protein values. Raw values can't tell you much until you do this step because the variation in growth between wells can be more than the difference in 2NBDG uptake between experimental groups. If you get good standard curves for both 2NBDG and on the protein assay, your final values for 2NBDG uptake will be in terms of mg of 2NBDG per mg of protein.

For my experiment, I was testing the effects of insulin (I'm not sure what you are testing or what your experimental groups are), which is present in serum, so I did an overnight serum starvation with 1%BSA instead of serum (at least 4 hours were needed) to get rid of any residual effects of insulin. I also found that both glucose starvation and high glucose before the experiment blunted 2NBDG uptake (at least in my cells). I would suggest low glucose concentrations leading up to the experiment if anything, but not a complete removal of glucose until you add 2NBDG. Also when I say "low glucose" I mean 5.5mM, the low glucose version of DMEM, which is actually what normal concentrations are in circulating blood. I found that high glucose DMEM (25mM) actually made my cells insulin resistant, and they needed at least a day in 5.5mM glucose beforehand.

One more thing that may help increase your raw values (and thereby increase the difference between experimental groups) is to make sure 485 and 535 are the optimal wavelengths. I know those wavelengths will work for 2NBDG, but I tested what the optimal excitation and emission wavelengths were on the plate reader before my experiments and found 465 and 540 to be optimal. It might be slightly different for every batch, so its a good idea to figure that out.

Good luck!

Thank you so much sir for the suggestions. Your suggestions are very meaningful.

Actually I was confused after seeing the values which are in pointers like0.8, 0.6 that whether my cells are uptaking 2-NBDG or not. I thought that the values should be like 3,5,6,8 around . Actually I have seen many papers in that they measure fluorescence in terms of MFU or AU which comes in 100 or 1000. But my values always come in pointers.

Yes i always runs negative control in my sample and then I substracted from the 2-NBDG values. As you said by giving an example that if after the difference between samples and auto fluorescence is 0.1 then it is 50% increase in fluorescence. Is it really so ? I am wondering whether the increase in only 0.1 is 50% as this increase may also be happen because of 5-10% difference in cell number of different samples.

As before going for fluorescence measurement should I quantify the amount of protein of each well?? I am not able to understand that how to plot a standard curve. Could you please elaborate?

should I need to check the optimal exitation and emission wavelength of 2-NBDG in my sample (2-NBDG) batch?

AU just means "arbitrary units", and I think MFU is "mean fluorescent units"... the values are still meaningful, but only when being compared to other values from that same experiment. You don't necessarily have to do a standard curve with 2NBDG, and its fine to have AU as your units, but you have to do the standard curve for the protein assay and then normalize your fluorescent values to total protein for each well.

Get a Pierce BCA protein assay kit. It will have detailed instructions on how to do a standard curve and the protein assay. In short, you load known amounts of protein (included in the kit) into a 96 well plate, with the concentration of protein doubling with each row (for example, 1st row = no protein, 2nd row = 10nM, 3rd row = 20nM, 4th row = 40nM, and so forth). After getting the values from the plate reader your results should look like a straight line through your data points. The slope of that line is the equation that will let you convert raw values from the plate reader into mG of total protein for a given well. Like I said, the kit will have better instructions than what I can write here. But getting total protein content for each well in order to normalize your fluorescent values is crucial. You don't have to do this step before, it can be done after. That part doesn't matter (in fact, after getting your fluorescent values, you can just put the plate in the freezer and do the protein assay the following day).

As far as subtracting autofluorescence values from experimental values, all I'm saying is the difference between raw values of 0.4 and 0.45 is only 0.05, which is a little over a 10% difference. However, if you have autofluorescent values of 0.3, subtract that from experimental values and you now have values of 0.1 and 0.15. The difference is still 0.05, but 0.05 out of 0.1 is 50%. At this point however, you don't know what that difference is due to, and you would have to do the protein assay and normalize your data first.

Determining the optimal excitation and emissions spectrum for the 2NBDG helped a little bit for me. 485/535 still worked, but 465 and 540 worked better. Your plate reader should have a function and instructions for this.

Good luck!

I followed Lyle Babcock's protocol and it works, thank you again for your help.

My values are 4935 for insuline + 2-NBDG and when I see your values Priya Goyal, it's very different from my results. All my values are around 1000 for my negative control and 4000 for positive one.

Ok sir. Thank you so much for your suggestions. I will do protein quantification also and then come back with the results.

Yes Dounia Hamoudi . Actually that Is why I am little bit confused whether my reading are correct or not.

I have read many papers and in that the value of fluorescence is in 3000 or 4000 and not like 0.4, 0.6. I actually don’t know the reason behind it whether It varies from instrument to instrument or there is some other reason.

Lyle Babcock , sir could you please explain.

Thank you.

I've used two different fluorescent plate readers, one gave readings anywhere from 3.0 to 10.0, the other (the one I used for my data on this project) gave readings of 0.3 to 1.0. I'm not exactly sure why there is a difference, I'm sure there is some setting on the software, but it also doesn't matter how your results are annotated as long as you explain it. I actually converted all my values to percent difference from the control group.

Okk sir. Thank you so much for the reply.

Thank you for the valuable information.

May I ask, how to evaluate the optical emission and excitation wavelength? What figure would let us know that the wavelengths are optimal or not?

The software for most fluorescent plate readers should have a way of measuring optimal excitation/emission wavelength (it'd be weird if one didn't). For me the optimal excitation was 468, and optimal emissions were 540.

I have a query regarding insulin stimulated glucose uptake that whether insulin and 2-NBDG should be incubated simultaneously for 1hr(or so) or first incubate the cells with insulin for 30min or so and then add 2-NBDG in the same insulin media and then incubate it for another hour.

One more query I am having is that insulin, recombinant human which I have purchased from sigma is in powder form. It gets soluble in MQwater at pH 2-3 and then stored at 4 degree. Is it remains stable at 2 or 3 pH at 4degree or we have to make up its pH at 7 to maintain its stability.

Thank You.

I found the optimal conditions to be 30 minutes in glucose/serum-free DMEM with both insulin (20nm) and 2NBDG (200um).

Hello sir

Have you added 2-NBDG and insulin simultaneously or first incubate the cells with 2-NBDG and then with insulin?

Simultaneously