Non-glycolytic acidification: last measure before glucose, or lowest ECAR after 2-DG?

I'm doing glycolysis stress tests,  and to calculate glycolysis, we are supposed to take the highest ECAR-value after glucose and subtract the ECAR-value representing the non-glycolytic acidification. In the classic examples from Agilent, the non-glycolytic acidification is given by the last measurement before glucose injection, but in our experiments most samples have a clearly lower ECAR after 2-DG than they have right before the glucose injection. This indicates that our cells have the ability to do some glycolysis at basal conditions, even if the assay medium doesn't contain any glucose (we have not starved the cells before the experiments - they are just incubated in the glucose-free assay medium for up to 1h before the analyses start). So: which value represents the true, non-glycolytic acidification; the last, basal measurement before glucose injection, or the lowest ECAR obtained after giving 2-DG which totally blocks glycolysis? I would expect the latter, but in the examples from Agilent they use the last measurement before glucose. How do others calculate this? The automatic report generator uses the last measure before glucosis, which I find strange IF the ECAR is lower after 2-DG...

  • Hi there,

    Thank you for contacting Cell Analysis Technical Support.  Unfortunately, it sounds like we will need further information to answer this question.  See our parameter equations below. Can you please send your Wave data file to 

    Thank you and I look forward to hearing from you,


  • Hi,

    I think you can consider this as a principle question (I don't have access to any data until tomorrow - it is late in the evening here). I'm aware that the «Rate Measurement Equation Used by Report Generator» states that «Non-Glycolytic Acidification» is given by «Last rate measurement prior to glucose injection». And in the typical figures presented as examples by Agilent, the ECAR value for this last rate measurement prior to glucose injection is comparable to the ECAR obtained after injecting 2-DG. In such a situation I have no problems with understanding that the non-glycolytic acidification can be calculated like Agilent recommends. However, in many of our glycolysis stress-tests, the ECAR obtained after 2-DG is clearly LOWER than the last rate measurement prior to glucose injection. This means that our cells actually do perform some glycolysis in the beginning of our experiments (perhaps due to some intracellular glucose still present). So, I would expect that it is more correct to use the lowest ECAR value obtained after the 2-DG injection as a measure of the non-glycolytic acidification since the cells now are unable to do any glycolysis. Was this understandable? 

    Sonja Slight smile

  • Hi Sonja,

    This is because that the cells have been starved of glucose at this point. If they have endogenous sources of glucose, they would have been utilizing them during the glucose starvation. If you have concerns of the role glycolysis fueled by internal fuels, you could try to adjust the length of starvation. 

    The measurements after 2-DG injection is not a good data point for non-glycolytic acidification for the following reasons,

    ·         Glucose is supplied from the first injection.

    ·         2-DG inhibits glucose response by competition. It does not completely block the glycolysis.

    I believe this answers your initial question, but please let me know if it does not!  I would also like to take a look at your data for verification purposes.

    Thank you!


  • Hi Sonja,

    I also meant to ask - what concentration of 2-DG are you using?

    Thank you,


  • Hi again,

    I have sent my question to and have got a reply from May Sanderhoff in Denmark. She has now got a copy of one of our protocols and a result file, and hopefully I will hear from her in a while. I think, however, it will be a waste of time to go through our experimental setup since my point is that this is a general question that could be answered independent of our specific results. It is a fact that the ECAR value could be lower after 2-DG than the baseline measurement before glucose injection, so I was hoping for an answer on a general basis. The report generator from Agilent seems to have as a default function that it uses the last baseline measurement to identify the non-glycolytic acidification, and I can't understand that this is correct for those cells/experiments where 2-DG causes a lower ECAR value. You mention that the 2-DG works by competition with glucose, but still the ECAR gets lower than the baseline measurements - even if the cells now, at the end of the assay, has an excess of glucose present. This should mean that the 2-DG is very effective in blocking the glycolysis, and that our cells actually have some glucose available intracellularly when we start the experiment and the baseline measurements take place (after 1h in assay medium without glucose - and I don't think we can keep there for any longer since they have a tendency to detach).

    We use 50 mM 2-DG, by the way.

    Even if I hopefully now will get some feedback via my email, I would still like to show you some figures to illustrate my point. I have tried to attach a pdf-file, but it didn't seem to work... I can send you a personal message (if I'm able to) and if this is OK with you.

    Best regards, Sonja

  • Hi Sonja,

    Thank you for the additional information!  My colleague, May, has let me know she is in contact you via email, so I will let you continue the discussion there.  Once you have an answer, I will be able to post it here.  Have a good day in the meantime!

    Thank you,


  • Update: The data was actually quite unusual because 2DG causes ECAR to drops so much. I know that our illustration in our user guides shows the same response to 2DG but what we usually see in real life is that 2DG does not make ECAR decrease to the same extent. This because 2DG is not an inhibitor but a competitor to glucose and therefore a tiny fraction of glucose is still metabolized. 2DG injection should only be considered as a control in this assay – to check that glycolysis can be stopped when glucose is ‘taken away’. This verifies that glucose is indeed the reason for the higher ECAR response. Therefore 2-DG cannot be used as the indicator for the non-glycolytic rate.

     Also the first 3 measurement cannot be considered as the basal ECAR as the cells have been starved and has no access to glucose. I therefore find the publication describing the basal a bit strange. You could say that basal ECAR is after the glucose injection and before oligomycin.

  • Hello, 

    I also had a lower ECAR after 2-DG, as described in this post by the SBA user.

    At baseline (medium without glucose or pyruvate) ECAR was at ~ 1 mpH/min/1,000 cells, whereas after 2-DG dropped to ~ 0.5 mpH/min/1,000 cells.

    Do you have any further input on this?

    Many thanks in advance!

  • Hi Eleanna,

    Thank you for contacting Cell Analysis Technical Support. For this case, we ask you to send your data file to so that I can take a look. It may be that there are not enough cells in the wells - have you done a cell density optimization yet?  I will be looking out for your data file!

    Thank you,



    Courtney Nadeau Watts

    Technical Support Scientist/Remote Engineer

    Cell Analysis Products

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