Agilent seahorse xfp analyzer

Metabolic analyses were performed in the Agilent Seahorse XFp Analyzer (Agilent Technologies). This instrument creates a transient micro-chamber of only a few microliters in specialized cell culture microplates which enables the oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), to be monitored in real time. shCTRL and shANK cells were seeded into a 6-well plate and incubated overnight in a humidified CO₂ incubator. Medium was then removed and replaced by fresh culture media with and without TRA (21 μg/mL). After 48 h of drug treatment, cells were plated in 8-well plates for 24 h (2 × 10⁵ cells per well) in complete media. Cells were changed to unbuffered DMEM (DMEM supplemented either 25 mM glucose, 1 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax, pH 7.4) and incubated at 37°C in a non-CO₂ incubator for 60 min. Three measurements were taken before and after sequential injection of mitochondrial inhibitors (1.0 μM oligomycin and 1.0 μM FCCP). OCR and ECAR were automatically calculated by Seahorse XFp software. Every point represents an average of n = 3.

OCR values were measured as described earlier (60 (link)), with some modifications, using unbuffered (pH 7.35) minimal medium (M. smegmatis) and Middlebrook 7H9 medium (M. tuberculosis) free from any carbon sources.
For the M. smegmatis experiments, a culture in the mid-log phase of growth (optical density at 600 nm [OD₆₀₀] of 0.8) was starved for 6 h in carbon source-free minimal medium containing 0.01% tyloxapol. Then, 2 × 10⁵ to 4 × 10⁵ cells/well were added to a Cell-Tak (Corning)-coated XF 96 cell culture microplate and were analyzed using an Agilent Seahorse XFe96 analyzer. OCR values were measured for about 21 min before the addition of preloaded ethanol (1%) or of medium through injection ports of the sensor cartridge. Finally, 5 mM CCCP was added to the wells and the measurement was continued for another 21 min.
For the M. tuberculosis experiments, a culture in the log phase of growth (OD₆₀₀ of 0.6 to 0.8) was starved for 18 to 24 h in carbon source-free 7H9 medium containing 0.01% tyloxapol. Subsequently, about 5 × 10⁵ cells/well were added to a Cell-Tak-coated XFp miniplate and analyzed using an Agilent Seahorse XFp analyzer. The assay conditions were similar to those described for M. smegmatis.

Mitochondrial respiration was measured using an Agilent Seahorse XFp analyzer (Agilent Seahorse, Santa Clara, CA, USA) and Seahorse XF Cell Mito Stress Test Kit (#103010-100). The test was performed following the manufacturer’s protocol. hESC-CMs were seeded on a 0.1% gelatin-coated XFp cell culture plate at 15,000 cells/well in RPMI containing 20% FBS and maintained for 3 days in RPMI/B27 medium. The day before the experiment, a sensor cartridge was hydrated in an XF calibrant at 37 °C in a non-CO₂ incubator. The XF assay medium was supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, and 10 mM D-(+)-galactose at pH 7.4 and 37 °C. One hour before the analysis, the hESC-CMs were replenished with XF assay medium after washing with XF medium. The cells were then incubated in a non-CO₂ incubator at 37 °C. Thirty minutes before the analysis, 2 μM oligomycin, 0.5 μM FCCP, and 2 μM each of a mix of antimycin A/rotenone was loaded into the A, B, and C injection ports of a hydrated sensor cartridge with 20 μL, 22 μL, and 25 μL of a 10× solution of components in the XF Mito Stress Test kit. After the calibration was completed, the cell culture plate was injected into the instrument.

The Agilent Seahorse XF technology was used to analyze the cell energy phenotype of HA and A172 cells using the Agilent Seahorse XFp Cell Energy Phenotype Test Kit (Agilent, 103275-100, Santa Clara, CA, USA) based on the instructions. HA and A172 cells were grown in the Agilent Seahorse XFp Cell Culture Miniplate and cultured overnight. The Agilent Seahorse XFp Sensor Cartridge was hydrated using Agilent Seahorse XF Calibrant at 37 °C in a non-CO₂ incubator overnight. The Agilent Seahorse Sensor Cartridge, Calibrant, and Miniplate were obtained from the Agilent Seahorse XFp FluxPak (Agilent, 103022-100). The assay medium was prepared by supplementing Agilent Seahorse XF Base Medium (Agilent, 102353-100) with 1 mM pyruvate (Sigma, S8636, St. Louis, MO, USA), 2 mM glutamine (Sigma, G8540), and 10 mM glucose (Sigma, G8769). The cell culture medium of HA and A172 were replaced with the assay medium and cultured in a non-CO₂ incubator for 1 h. Oligomycin and cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP) from the Agilent Seahorse XFp Cell Energy Phenotype Test Kit were combined to develop a stressor mix loaded into every port A of the hydrated sensor cartridge. The Agilent Seahorse XF Cell Energy Phenotype test was run using the Agilent Seahorse XFp Analyzer (Agilent,102745-100). The data were analyzed with the Agilent Seahorse XF Cell Energy Phenotype Test Report Generator.