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

Question

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...

Courtney Watts · Accepted Answer

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 &ndash; to check that glycolysis can be stopped when glucose is &lsquo;taken away&rsquo;. 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.

Eleanna · Answer

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!

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

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