Oxygraphy was performed as previously described [23 (link),24 (link)] with a technical n of ≥ 3. Briefly, cells cultured and harvested as above from a T175 were resuspended in Miro5 buffer to 20 million cells/mL and 100 μL (2 million cells) were injected per chamber of the oxygraph, with subsequent injections of the following compounds to the final concentration of: digitonin, (Merck) 7.5 µg/mL as determined by a digitonin titration; pyruvate, 5 mM; malate, 0.5 mM; ADP (Calbiochem), 1 mM; glutamate, 10 mM; succinate, 10 mM; carbonyl cyanide m-chlorophenyl hydrazone (CCCP), Δ0.5 µM until maximum respiration reached; rotenone, 75 nM; glycerophosphate, 10 mM; antimycin A, 250 nM; ascorbate sodium salt (Merck), 2 mM; N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD), 0.5 mM; sodium azide, 200 mM. Oxygen saturation was maintained at ≥ 20 µM throughout the experiment, and at ≥ 180–200 µM before the measurement of isolated complex IV (CIV) activity.
Ascorbate sodium salt
Manufactured by Merck Group
Ascorbate sodium salt is a water-soluble chemical compound. It serves as a source of the vitamin C (ascorbic acid) in laboratory and research applications.
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Lab products found in correlation
2 protocols using ascorbate sodium salt
1
Measuring Cellular Respiration and Complex IV Activity
2
Oxygraphy of Fibroblasts: Mitochondrial Respiration
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Oxygraphy was performed as described previously [11 ] using the Oxygraph O2K from Oroboros instruments accordingly to previously published resources with a technical n of ≥3 [11 ,14 (link)]. Briefly, fibroblasts were harvested by trypsinisation from a T175 flask at a density of 80%, washed in PBS (phosphate buffered saline) and resuspended in Miro5 buffer to 20 million cells/mL. Two million cells (100 μL) were injected per oxygraph chamber. Subsequently, the following compounds were injected to the final concentration of: digitonin, (Merck) 7.5 μg/mL as determined by a digitonin titration; pyruvate, 5 mM; malate, 0.5 mM; ADP (Calbiochem), 1 mM; glutamate, 10 mM; succinate, 10 mM; carbonyl cyanide m-chlorophenyl hydrazone (CCCP), Δ 0.5 μM till maximum respiration reached; rotenone, 75 nM; glycerophosphate, 10 mM; antimycin A, 250 nM; ascorbate sodium salt (Merck), 2 mM; N,N,N′,N'-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD), 0.5 mM; sodium azide, 200 mM. Oxygen saturation was maintained at ≥ 40 μM throughout the experiment, and at ≥ 180 μM before the measurement of isolated complex IV (CIV) activity.
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