The XFe Extracellular Flux Analyzer (Agilent Technologies, Inc.) was used to detect the ECAR and OCR in the present study. The ECAR was detected using an XF Glycolysis Stress Test kit (Agilent Technologies. Inc.), and the OCR was detected using an XF Cell Mito Stress Test kit. A total of 1×104 MG63 and Saos-2 cells were seeded in matched 96-well microplates. Following baseline measurements, 30 mM glucose, 2 µM oligomycin (an inhibitor of oxidative phosphorylation; Selleck Chemicals) and 10 mM 2-DG (an inhibitor of glycolysis; Selleck Chemicals) were added to each well at the specified time points to detect the ECAR. Similarly, 2 µM oligomycin, 5 µM FCCP (a reversible inhibitor of oxidative phosphorylation; Selleck Chemicals) and 1 µM rotenone (an inhibitor of mitochondrial complex I; Selleck Chemicals) plus 1 µM antimycin A (an inhibitor of mitochondrial complex III; Selleck Chemicals) were added in the appropriate order to detect the OCR and ECAR according to the operating steps described in a previous study (20 (link)). The data were collected and analyzed using XF-96 Wave software version 2.1 (Agilent Technologies. Inc.). The data were normalized based on the protein concentration.
Protocol detail
Extracellular Metabolic Profiling of Osteosarcoma Cells
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XFe Extracellular Flux Analyzer
XF Glycolysis Stress Test kit
XF Cell Mito Stress Test kit
oligomycin
FCCP
rotenone
antimycin A
Antimycin a
Cells
Complex iii
Fccp
Glucose
Glycolysis
Mito
Mitochondrial
Mitochondrial complex i
Oligomycin
Oxidative phosphorylation
Protein
Rotenone
Saos
Stress test
Corresponding Organization :
Other organizations : Guizhou Provincial People's Hospital
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Variable analysis
independent variables
- Addition of 30 mM glucose
- Addition of 2 µM oligomycin
- Addition of 10 mM 2-DG
- ECAR (Extracellular Acidification Rate)
- OCR (Oxygen Consumption Rate)
- Seeding of 1×10^4 MG63 and Saos-2 cells in matched 96-well microplates
- 2 µM oligomycin (an inhibitor of oxidative phosphorylation)
- 5 µM FCCP (a reversible inhibitor of oxidative phosphorylation)
- 1 µM rotenone (an inhibitor of mitochondrial complex I)
- 1 µM antimycin A (an inhibitor of mitochondrial complex III)
- No information provided
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