Measuring Cellular Bioenergetics by Seahorse

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)].

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Lang L., Loveless R., Dou J., Lam T., Chen A., Wang F., Sun L., Juarez J., Qin Z.S., Saba N.F., Shay C, & Teng Y. (2022). ATAD3A mediates activation of RAS-independent mitochondrial ERK1/2 signaling, favoring head and neck cancer development. Journal of Experimental & Clinical Cancer Research : CR, 41, 43.

Publication 2022

Seahorse XF Cell Energy Phenotype Test Kit Seahorse XFe96 Extracellular Flux analyzer

Cancer Carbonyl cyanide phenylhydrazone Cells Glucose Glutamine Lysine Oligomycin Oxygen consumption Phenotype Poly Pyruvate Seahorse Sodium

Corresponding Organization : Emory University

Other organizations : The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology

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Variable analysis

independent variables

Oligomycin (Oligo) concentration (1 μM)
Fluoro-carbonyl-cyanide phenylhydrazone (FCCP) concentration (1 μM)

dependent variables

Oxygen consumption rate (OCR)
Extracellular acidification rate (ECAR)
OCR:ECAR ratio

control variables

Cell density (4 × 10^3 cells per well)
Culture medium (XF medium with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate)
Incubation time (45 minutes)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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