The Mito Stress Test Kit (Agilent, 103015-100), Glycolytic Rate Assay Kit (Agilent, 103344-100), and ATP Rate Assay Kit (Agilent, 103592-100) were used to evaluate the oxygen consumption rate (OCR), glycolytic proton efflux rate (GlycoPER), and ATP production rates through the Seahorse Bioscience XF96 extracellular flux analyzer (Agilent Technologies), respectively. Cells were seeded into the Seahorse XF 96-well culture plates (10,000 cells per well) and maintained in complete culture medium overnight. Then the cells were incubated with BD under hypoxia for 24 h, and the cell culture medium was replaced with bicarbonate-free low-buffered assay medium (supplement with 10 mmol/L glucose, 1 mmol/L pyruvate, 2 mmol/L glutamine, and 5 mmol/L HEPES) in a 37 °C CO2-free incubator for 1 h. After base-line measurement, oligomycin, FCCP, and rotenone/antimycin A were added for the detection of OCR value. Similarly, rotenone/antimycin A and 2-DG (inhibitor of glycolysis) were added at the time points specified according to the manufacturer's protocols for the measurement of GlycoPER value. Also, the ATP production rate of mitochondrial oxidative phosphorylation and glycolysis was measured in the presence of oligomycin and rotenone/antimycin A.
Atp rate assay kit
Manufactured by Agilent Technologies
The ATP Rate Assay Kit is a laboratory tool used to measure the rate of adenosine triphosphate (ATP) production or consumption in biological samples. It provides a quantitative assessment of ATP levels, which is a key indicator of cellular energy metabolism.
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Lab products found in correlation
Mito stress test kit, by Agilent Technologies (2 mentions)
Seahorse bioscience xf 96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Glycolytic rate assay kit, by Agilent Technologies (1 mentions)
Seahorse xf real time atp rate assay, by Agilent Technologies (1 mentions)
Wave software, by Agilent Technologies (1 mentions)
Sodium pyruvate, by Agilent Technologies (1 mentions)
Agilent seahorse xf cell energy phenotype test kit, by Agilent Technologies (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
3 protocols using atp rate assay kit
1
Evaluating Cellular Bioenergetics via Seahorse
2
Real-Time Cellular ATP Kinetics Assay
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The Agilent Seahorse XF Real-Time ATP Rate Assay was conducted following the manufacturer’s recommendations using the ATP rate assay kit (Agilent, Catalog # 103592-100). BMDMs were isolated and plated, then treated with LPS as described above. The Seahorse media for the ATP rate assay was prepared according to the manufacturer’s instructions the day before the assay and supplemented with 15 mM glucose (Sigma), 4 mM glutamine (Gemini) and 1 mM sodium pyruvate (Agilent). The assay was conducted according to the manufacturer’s protocol. The final concentrations of the injection compounds used in this assay were oligomycin (1 µM) and rotenone/antimycin A (0.5 µM). At the end of the assays, the cells were collected in RIPA buffer for protein estimation. The data were collected and analyzed using Wave software (Agilent).
3
Evaluating Cellular Metabolic Profiles
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Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using the Agilent Seahorse XFe96 Analyzer with fibroblasts from the healthy volunteer and the patients carrying the mutations.
The cells were seeded in Agilent Seahorse 96-well XF cell culture microplates at a density of 4 × 104 cells per well in 180 μL of growth medium, and were allowed to adhere for 24 h in a 37 °C humidified incubator with 5% CO2. Before running the assay, the Seahorse XF Sensor Cartridge was hydrated and calibrated with 200 μL of Seahorse XF Calibrant Solution in a non-CO2 37 °C incubator to remove CO2 from the media that would otherwise interfere with the pH-sensitive measurements. Subsequently, the Agilent Seahorse XF Cell Energy Phenotype Test Kit, Mito Stress Test Kit, ATP Rate Assay Kit and Glycolytic Rate Assay Kit and Glycolysis Stress Test were performed according to manufacturer protocols.
All kits and reagents were purchased from Agilent Technologies (Santa Clara, CA, USA).
The cells were seeded in Agilent Seahorse 96-well XF cell culture microplates at a density of 4 × 104 cells per well in 180 μL of growth medium, and were allowed to adhere for 24 h in a 37 °C humidified incubator with 5% CO2. Before running the assay, the Seahorse XF Sensor Cartridge was hydrated and calibrated with 200 μL of Seahorse XF Calibrant Solution in a non-CO2 37 °C incubator to remove CO2 from the media that would otherwise interfere with the pH-sensitive measurements. Subsequently, the Agilent Seahorse XF Cell Energy Phenotype Test Kit, Mito Stress Test Kit, ATP Rate Assay Kit and Glycolytic Rate Assay Kit and Glycolysis Stress Test were performed according to manufacturer protocols.
All kits and reagents were purchased from Agilent Technologies (Santa Clara, CA, USA).
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