Mitochondrial stress test

Mitochondrial bioenergetics were evaluated by the use of the Seahorse 96-well plate reader. Cells were grown in specialty plates from the company, at 20,000 cells per well of N2A mouse neuronal cells. The cells were treated with compounds at 20 μM for three hours, and then a mitochondrial stress test (Seahorse Biosciences) done using compounds which affect mitochondrial function, including oligomycin, FCCP, rotenone, and antimycin A.

OXidative PHOSphorylation (OXPHOS) and glycolysis in MIA PaCa-2 cells were monitored in real time using the extracellular flux (XF) bioanalyzer XFe96 platform (Agilent, Santa Clara, CA, USA). Cells were cultured overnight in custom XF microplates with DMEM complete medium. Prior to measurements, cells were washed and incubated in unbuffered assay medium (Sigma-Aldrich, St. Louis, MO, USA) in the absence of CO₂ for 1 h at 37°C. The rates of mitochondrial respiration and glycolysis were measured using the mitochondrial stress test (Agilent, Santa Clara, CA, USA) and displayed as the Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR), respectively (Dranka et al., 2011 (link)). All measurements commenced after establishment of a stable baseline, followed by sequential addition of the LDH inhibitor NCI-006, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and finally antimycin A and 2-deoxyglucose.

HUVEC and HAEC were assayed using the Seahorse XF 96 extraflux analyzer (Agilent Technologies, Lexington, MA) according to manufacturer’s instructions. Briefly the cells were plated at 10,000 cells/well and allowed to adhere overnight. The next day, the cells were pretreated for 2h with 0, 20 or 100 µg/ml TXA (Mylan Technologies, St Albans city, VT) in full cell culture medium. Then, the medium was changed over to Seahorse XF DMEM base medium, without phenol red (Agilent, cat # 103335–100) supplemented with 10mM Glucose, 2mM Glutamine and 1mM Sodium Pyruvate pH 7.4, and the same TXA concentration as during the pretreatment, and then metabolic parameters were measured using the Mitochondrial Stress Test (Agilent, Cat # 103015–100). Analysis of oxygen consumption rate (OCR) was done using the following protocol after calibration and equilibration: 3 cycles of mix 3 min, wait 2 min, measure 3 min, starting with basal values, and then after each of the following injections: 1.25µM Oligomycin (final concentration), 1µM FCCP (final), and 0.5µm Rotenone and Antimycin (final). Post assay wells were washed, lysed, and a standard BSA assay was performed for protein content. Data presented are normalized for protein content/well and are representative of 3 independent experiments with at least 10 replicate wells/treatment within each experiment.