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...