The experiment was performed according to the instructions of the XF Glycolytic Rate Assay Kit instructions (Agilent, Santa Clara, CA, USA). Briefly, LO2 cells were plated at 15,000 cells/well in 96-well Seahorse XF 96 assay plates overnight before exposure to FFA (1 mM) and drug treatment for 24 h. The extracellular acidification rate (ECAR) was measured using an XF 96 flux analyser (Agilent, Santa Clara, CA, USA). On the day of this assay, the culture medium was changed to unbuffered DMEM (DMEM supplemented with 25 mM glucose and 10 mM sodium pyruvate; pH 7.4) and the cells were incubated at 37 °C in a non-CO2 incubator for 1 h. LO2 cells were stimulated with rotenone, antimycin A (0.5 μM) and 2-DG (50 mM). Eight independent experiments were performed. ECAR was automatically calculated and recorded using Seahorse XFe-96 software.
Xf96 flux analyser
Manufactured by Agilent Technologies
Sourced in United States
The XF96 Flux Analyser is a lab equipment product manufactured by Agilent Technologies. It is designed to measure and analyze various flux parameters in a controlled laboratory environment. The core function of the XF96 Flux Analyser is to provide accurate and reliable data on flux-related characteristics without interpretation or extrapolation.
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5 protocols using xf96 flux analyser
1
Extracellular Acidification Rate Assay
2
Neutrophil Metabolic Profiling via Seahorse XF96
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Seahorse XF96 Flux Analyser (Agilent) was applied to measure extracellular acid ratio (ECAR) according to the manufacturer’s instructions. Briefly, neutrophils were stimulated with AP serum for 6 h and then harvested. For ECAR test, 1 μmol/L oligomycin, 0.5 μmol/L carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 μmol/L rotenone plus 0.1 μmol/L antimycin A were injected to the wells.
3
Cellular Bioenergetic Profiling Using Seahorse XF96
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The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the cells was assessed using the Seahorse XF96 Flux Analyser (Seahorse Bioscience, Agilent). In brief, 1 × 104 cancer cells were seeded in an incubation plate as the protocol indicated. Cells were cultured at 37 °C overnight for adhesion. Before detection, culture medium was replaced with assay media. The glycolytic stress test kit (Seahorse Cat. #103020-100) and mitochondrial stress test kit (Seahorse Cat. #103015-100) were purchased for ECAR and OCR detection, respectively. The assays were performed using the manufacturer protocols.
4
Seahorse XF96 Flux Analysis Protocol
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Seahorse XF96 Flux Analyser (Seahorse Bioscience) was used to measure in vitro metabolic alterations. Cells were seeded at 2 × 104 per well in a XF-96-wll plate and cultured under a serum starvation condition for 24 h. Then cells were incubated in unbuffered medium, followed by a sequential injection of 10 mM glucose, 1 mM oligomycin (Sigma-Aldrich), and 80 mM 2-deoxyglucose (2-DG, Sigma-Aldrich, D8375). The measurements were normalized using the 0 h data as a standard. The concentration of BAPN was 200 µM.
5
Bioenergetic Analysis of CML c-Kit+ Cells
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BMDM were plated at 50,000 cells per well of a 96-well plate and allowed to adhere overnight. Wild-type and CML c-Kit+ cells were obtained as described above in c-Kit isolation and 10,000 cells per well were plated directly on top of macrophages, culturing both in mouse SFM for 24 h. Following co-culture, c-Kit cells were removed by washing 3× in PBS. Minimal DMEM was prepared with glucose (25 mM) and glutamine (4 mM), pH adjusted to 7.4, and 175 μL added per well. A mitostress test was performed using XF96 flux analyser (Seahorse Bioscience). according to manufacturer’s instructions. Injection drugs were prepared as 1.5 μM oligomycin, 1.5 μM CCCP, 1.25 μM rotenone and 2.5 μM antimycin A. Immediately after completion of the assay, protein extraction was performed, and measurements normalised to protein content of each well.
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