Xfp cell culture miniplate

Approximately 1 × 10⁵ cells of BMMs were plated in XFp Cell Culture Miniplate (Agilent Technologies, USA) overnight in BMMs media. The media were replaced with fresh DMEM complete medium and the cells were rested for 1 h before stimulation with BG (50 μg/mL) for 24 h. After resting for 48 h, glycolytic function was detected using Agilent Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, USA) following the manufacturer’s protocol. The glycolysis signal was measured by extracellular acidification rate (ECAR) by Agilent Seahorse XFp Analyzer (Agilent Technologies, USA). In brief, BMMs were incubated in glucose free assay medium and non-glycolysis acidification was measured. This step was followed by addition of glucose to the system (final concentration = 10 mM) to induce glycolysis function. Oligomycin, an ATP synthase inhibitor (final concentration = 50 mM) was added to inhibit the mitochondria function leading to induce the maximum glycolysis. Finally, 2-DG, a glycolysis inhibitor (final concentration = 500 mM) was added to confirm that ECAR signal in this system represents the glycolytic function. The data were analyzed using Agilent Seahorse Wave (Agilent Technologies, USA).

MiaPaca2 and PANC-1 cells were seeded at 10,000 cells/well in complete normal DMEM (25 mM Glucose and 2 mM Glutamine) in an Agilent XFp Cell Culture miniplate (#103025–100) at 37°C in 5% humidified CO₂ incubators, as recommended. OCR was performed using the XFp mini extracellular analyzer (Seahorse Bioscience). For OCR, cells were treated in the absence or presence of 5-FU under indicated glucose concentration for 36 hours. The XFp FluxPak cartridge (#103022–100), was hydrated with 200 μL/well of XF Calibrant (#100840–000) and incubated at 37 °C using non-CO₂ incubator overnight. The following day cells were washed and replaced with Seahorse XF base media (at indicated glucose concentrations), and the plate was incubated in a non-CO2 incubator at 37°C. The Mito Stress test kit (#103015–100) was reconstituted in an assay medium to make the following concentration of inhibitors: 1.5 μM oligomycin, 2.5 μM FCCP, and 0.5 μM rotenone + 0.5 μM antimycin A. OCR was measured using Wave 2.6 software, according to the manufacturer’s instructions, all data was normalized to cell number with Quant-iT PicoGreen^™ dsDNA Assay Kit (Invitrogen).

To evaluate mitochondrial respiration, the real-time ATP rate assay kit (Agilent, 103591-100) was employed in accordance with the manufacturer’s instructions. Real-time ATP production and oxygen consumption rate (OCR) were monitored with the aid of Seahorse XFp analyzer (Agilent). Cells at 90% confluency were plated on a XFp cell culture miniplate (Agilent, 1030250) in complete medium. The following day, cells were treated with XF DMEM, pH 7.4 (Agilent, 103575-100). Assay cycles involving 3 minutes of mixing and a 2-minute waiting period were conducted in triplicate. Following measurement of basal respiration, 1.5 μM oligomycin (75351, Sigma-Aldrich) and 1 μM rotenone/antimycin A mix (R8875 and A8674, Sigma-Aldrich) were added and measured.

The seahorse XFp Analyzer was used to perform metabolic analysis of ethanol (25 mM, 24 h) and LPS (20 ng/mL, 24 h) treated the RAW 264.7 murine macrophage after anti-miR-34a or control anti-miRNA transfections. The RAW 264.7 murine macrophage (1×10⁵ cells/well) was plated on an XFp Cell Culture miniplate (Agilent Technologies) and treated as specified. Oxygen consumption rate (OCR) was assessed using the XFp Analyzer (Seahorse Bioscience) following manufacture’s guidelines.