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.
Xfe96 platform
Manufactured by Agilent Technologies
Sourced in United States
The XFe96 platform is a high-performance metabolic analyzer designed for comprehensive analysis of cellular metabolism. It provides real-time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to assess mitochondrial function and glycolytic activity in live cells.
Automatically generated - may contain errors
3 protocols using xfe96 platform
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
Mitochondrial Respiration of Monocyte-Derived Macrophages
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Monocytes were plated initially onto a Seahorse 96-well plate at 50,000 cells per well and differentiated into MDM directly in the wells for 7 days as above. At that point, differentiation medium was removed and CFTR modulators were added for 48 h. Pa conditioned medium was prepared in advance by culturing bacteria overnight in Seahorse mitostress assay medium, normalizing cultures to an OD600 of 0.500, centrifuging to pellet bacteria then passing supernatant through a 0.22 µm filter. This was defined as 100% conditioned medium. Aliquots were then frozen at − 20 °C until use.
On the day of assay, cell culture medium was exchanged for mitostress assay medium as per manufacturer’s instructions. A mitostress assay with acute injection was run on an Agilent Seahorse XFe96 platform, with 20 µL Pa conditioned medium in injection port A (final concentration 10% after injection), plain mitostress medium was included for control wells. Oligomycin, FCCP, and Rotenone/Antimycin A were placed into ports B-D respectively as per mitostress protocol. 4–8 technical replicates per condition were run. Parameters including response to injection, maximal respiration, and others were calculated as per manufacturer’s instructions.
On the day of assay, cell culture medium was exchanged for mitostress assay medium as per manufacturer’s instructions. A mitostress assay with acute injection was run on an Agilent Seahorse XFe96 platform, with 20 µL Pa conditioned medium in injection port A (final concentration 10% after injection), plain mitostress medium was included for control wells. Oligomycin, FCCP, and Rotenone/Antimycin A were placed into ports B-D respectively as per mitostress protocol. 4–8 technical replicates per condition were run. Parameters including response to injection, maximal respiration, and others were calculated as per manufacturer’s instructions.
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