Seahorse xf cell energy phenotype test kit

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF96 analyzer (Seahorse Biosciences, North Billerica, MA, USA). Transfected and non-transfected murine MSC were plated on 96-well plates 6 h before the experiment in XF media (non-buffered DMEM medium, containing 5 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. OCR and ECAR were measured under basal conditions and in response to 25 mM D-Glucose, 2 μM of oligomycin, 2 μM of carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and 0.5 μM of antimycin A and rotenone (Seahorse XF Cell Energy Phenotype Test Kit from Agilent).). Three successive readings were taken after each sequential injection. The instrumental background was measured in separate control wells using the same conditions without biologic material. After the seahorse experiment, the plated cells were fixed for 10 min in 4% PFA and then, stained with HOESCHT for 5 min (1/8000e) to count cells with Agilent BioTek Cytation for normalization.

Live cell analyses of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by Seahorse XF Cell Energy Phenotype Test Kit on a Seahorse XFe96 Extracellular Flux analyzer (Agilent Technology) as previously described [27 (link)]. In brief, 4 × 10³ cancer cells were seeded on each well of poly-D-lysine coated XF96 miniplates overnight and then incubated in 100 μl XF medium (10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) for 45 mins. Three measurements were obtained under basal conditions and upon sequential injection of 1 μM oligomycin (Oligo) and 1 μM fluoro-carbonyl-cyanide phenylhydrazone (FCCP). OCR and ECAR values were calculated from 3-min measurement cycles and adjusted to cell numbers. OCR:ECAR ratio was calculated according to previous report [28 (link)].