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.
Unbuffered assay medium
Manufactured by Merck Group
Sourced in United States
Unbuffered assay medium is a laboratory reagent used to create a controlled environment for conducting various types of assays. It provides a balanced chemical composition without the addition of buffering agents, allowing for uninterrupted measurement and observation of the target analytes or reactions.
Automatically generated - may contain errors
3 protocols using unbuffered assay medium
1
Real-Time Monitoring of OXPHOS and Glycolysis
2
Real-Time Monitoring of OXPHOS and Glycolysis
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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.
3
Extracellular Acidification Assay for hESC-ECs
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After the indicated treatment, hESC-ECs were seeded at a density of 6 × 104 cells per well in sextuplicate into a gelatin-coated Seahorse XF24 culture plate. The extracellular acidification rate (ECAR) was measured the next day with a Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, USA) to indicate glycolytic flux as previously described [20 (link)]. Before measurement, cells were incubated in unbuffered assay medium (Sigma, USA) for 1 h in a non-CO2 incubator at 37 °C. ECAR was measured for five cycles. Each cycle consisted of a 3-min medium mixing time, a 2-min waiting time and a 2-min measurement time. Measurements were taken at four successive timepoints: before the addition of glucose (10 mM), before the addition of oligomycin (3 µM) and before and after the addition of the glycolysis inhibitor 2-DG (100 mM). All compounds were prepared in assay medium and adjusted to pH 7.4. ECAR values were normalized to the protein content.
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