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Ascorbate measurement For neutrophil ascorbate measurement, 10 l of the extraction solvent (80% methanol containing 10 mM EDTA and 50 pmoles 13 C-ascorbate/200,000 cells as an internal standard) was added to 10 L neutrophil extract, vortexed for 10, centrifuged at 21,000 g for 15 min at 4 C, the supernatant was diluted using 30 L of 0.03% formic acid in water and analyzed using an AB Sciex 6500+ QTrap mass spectrometer coupled to a Shimadzu UHPLC system operating in negative mode to detect the transitions 175/115 (ascorbate) and 176/116 ( 13 C-ascorbate)

Cellular effects measured over months

383,384 The secondary mtDNA depletion may exacerbate OXPHOS disturbance, increasing the production of mtROS