Mitochondrial Bioenergetics of Mesenchymal Stem Cells

Preliminary studies of MSC bioenergetics were used to determine that a seeding density of 30,000 MSCs/well produced optimal (100 pmol/min) basal OCR rates best suited for performing mitochondrial stress tests (Fig. 7B). Therefore, 30,000 MSCs were seeded per well into Seahorse™ XF96 Cell Culture Microplates (Agilent) and allowed to adhere for up to 24 h on the day before the assay. On the day of the assay, MSCs were washed once and incubated in DMEM supplemented with 10 mM glucose and 2 mM L-glutamine. After calibration of the Seahorse XF96 Analyzer, the Seahorse™ XF96 Cell Culture Microplate was inserted and basal oxygen consumption rates (OCR) were determined followed by further OCR measurements after the addition of mitochondrial inhibitors Oligomycin (1 µM), carbonyl cyanide-p-triflouromethoxyphenylhydrazone (FCCP, 1 µM), rotenone/antimycin ( 0.5 µM/1 µM) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)/ascorbic acid (0.5 mM/2 mM) as described previously^{8 (link),57 (link)}.