Mitochondrial Respiration and Metabolism Assays

Mitochondrial fractions were prepared from cultured cells as we previously described [72 (link)]. Mitochondrial respiration assays using freshly isolated mitochondria were performed by measuring O₂ consumption in a 2-channel respirometer (Oxygraph-2k with DatLab software; Oroboros Instruments, Innsbruck, Austria) as we previously described [10 (link), 11 (link), 15 (link), 72 (link)]. Glucose uptake experiments were carried out as previously described [10 (link), 11 (link)] using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Life Technologies, Grand Island, NY; catalog # N13195). Lactate accumulation in the culture medium was determined using a fluorescence-based L-lactate assay kit from Cayman (Ann Arbor, MI) according to the manufacturer's instructions. Mitochondrial complex activities were determined, with results normalized to citrate synthase activity, as previously described [10 (link), 55 (link), 73 (link)].

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Oliva C.R., Ali M.Y., Flor S, & Griguer C.E. (2022). COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM. Cell Stress, 6(4), 45-60.

Publication 2022

Oxygraph-2k DatLab software 2-NBDG L-lactate assay kit

2 deoxyglucose 2 nbdg Assay Cayman Citrate synthase Culture medium Cultured cells Fluorescence Glucose Lactate Mitochondrial Respiration

Corresponding Organization : University of Iowa

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Variable analysis

independent variables

Mitochondrial respiration assays using freshly isolated mitochondria

dependent variables

O2 consumption
Glucose uptake
Lactate accumulation in the culture medium
Mitochondrial complex activities

control variables

Mitochondrial fractions were prepared from cultured cells as previously described [72]
Mitochondrial respiration assays were performed by measuring O2 consumption in a 2-channel respirometer (Oxygraph-2k with DatLab software; Oroboros Instruments, Innsbruck, Austria) as previously described [10, 11, 15, 72]
Glucose uptake experiments were carried out as previously described [10, 11] using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Life Technologies, Grand Island, NY; catalog # N13195)
Lactate accumulation in the culture medium was determined using a fluorescence-based L-lactate assay kit from Cayman (Ann Arbor, MI) according to the manufacturer's instructions
Mitochondrial complex activities were determined, with results normalized to citrate synthase activity, as previously described [10, 55, 73]

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