To measure the extracellular acidification rate (ECAR), we used a Seahorse extracellular flux (XF96) analyzer. In all, a total of 2 × 104 cells were cultured in XF96 Cell Culture Microplate pre-coated with Matrigel in mES medium before 24 h from the assay. After a medium change to XF base media supplemented l -glutamine (4 mM, Gibco, 25030–081), the assay was performed using XF96 Extracellular Flux Analyzers (Seahorse Bioscience, North Billerica, MA, USA). Four measurements were obtained under basal conditions and after the addition of several chemicals such as d -glucose (10 mM), oligomycin (1 μM), and 2-Deoxy-d -glucose (50 mM) (Agilent Technologies, 103020–100). The process after treatment was performed according to the manufacturer's instructions.
Extracellular flux xf 96 analyzer
Manufactured by Agilent Technologies
Sourced in United States
The Extracellular Flux (XF-96) analyzer is a laboratory instrument designed to measure the metabolic activity of cells. It provides real-time, noninvasive measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a multiwell plate format.
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Lab products found in correlation
4 protocols using extracellular flux xf 96 analyzer
1
Extracellular Acidification Rate Measurement
2
Glycolysis Profiling of Glioma Cells
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Glycolysis and glycolytic capacities were determined for U87 and U251 cells using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience, Billerica, MA) [22 ]. Cells were seeded in XF96-well plates and incubated at 37°C in a 5% CO2 humidified atmosphere for 24 h. Extracellular acidification rates (ECARs) were simultaneously measured real time after 1 d in XF-96. Initially, the cells were incubated in the glycolysis stress test medium without glucose, and ECARs were assessed. Three sequential injections of D-glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (100 mM) were injected in turn, and ECARs were assessed. Non-glycolytic acidification was defined as initial and final ECARs. Glycolysis was defined as ECAR following addition of D-glucose and maximum glycolytic capacity, which was defined as ECAR following addition of oligomycin.
3
Mitochondrial Respiration Profiling
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To measure oxygen consumption rate (OCR), an indicator of mitochondrial respiration, we used the state-of-the-art Seahorse Extracellular Flux (XF) 96 Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Basal respiration, norepinephrine-induced respiration, ATP-linked respiration, proton leak respiration and reserve capacity were determined for each parameter in three repeated rates over a 20 min period. Baseline cellular oxygen consumption was measured by subtracting non-mitochondrial respiration.
4
Glycolytic Capacity Measurement in PC3M and PCS Cells
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Glycolysis and glycolytic capacity were determined for PC3M and PCS cells using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience, Chicopee, MA). Cells were cultured for 2 hours in the absence of glucose. Three sequential injections of D-glucose (2 g/L), oligomycin (1 μM), and 2-Deoxyglucose (100 mM) provided extracellular acdification (ECAR) associated with glycolysis, the maxiumum glycolytic capacity, and non-glycolytic ECAR. Glycolysis was defined as ECAR following the addition of D-glucose and maximum glycolytic capacity was defined as ECAR following the addition oligomycin. ECAR following treatment with 2-Deoxyglucose is associated with non-glycolytic activity.
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