4.9. Seahorse Analysis

The cellular bioenergetics were determined using the XFp analyser (Agilent, Boston, MA, USA) kindly provided by Perlan Technologies Poland. All assays were programmed (designed) in XF data acquisition Wave 2.6.1 software (Agilent, Boston, MA, USA). In each experiment, 3 baseline measurements were taken prior to the addition of any compound/substrate/inhibitor, and at least 3 response measurements were taken after the addition of each compound. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were reported as absolute rates (pmoles/min for OCR and mpH/min for ECAR). While sensor cartridges were hydrated (overnight) and calibrated (XF Calibrant), cell plates were incubated in a 37 °C for 30 min prior to the start of an assay. All experiments were performed at 37 °C in non-CO₂ conditions. Detailed protocols and their justification can be found at https://www.agilent.com/en/product/cell-analysis/how-to-run-an-assay (accessed from June 2020 to May 2021). Additionally, detailed protocols were previously published [103,104].

Neutrophils were suspended in sterile (0.2 um syringe strainer filtered) HBSS(+) w/o sodium bicarbonate (Gibco, Waltham, MA, USA) supplemented with 1 mM sodium pyruvate (Sigma–Aldrich, Saint Louis, MO, USA), 2 mM L-Glutamine (Sigma–Aldrich, Saint Louis, MO, USA), 10 mM D-glucose (Lonza Bioscience, Basel, Switzerland) and 5 mM HEPES (Sigma–Aldrich, Saint Louis, MO, USA) and adjusted to pH 7.4 with 0.1 N NaOH (Sigma–Aldrich, Saint Louis, MO, USA). Buffer factor of assay media was validated prior to experiments and was equal to 2,9 mM/pH. Cells were plated (300,000 cells/well) in 180 μL on Agilent Seahorse 8-well XFp Cell Culture Miniplate and allowed to settle/adhere for 30 min at 37 °C. Real-time, noninvasive measurements of ECAR and OCR were obtained which correlated to acidification, mostly derived from glycolysis and mitochondrial function, respectively. While the assay was running, at 12 min of baseline measurement, acute injection of LPS suspended in assay medium (75 µg/mL final well conc., port A, 20 μL) was done, and measurements were continued for 1 h. Measurements taken after the LPS injection consisted of (i) a sample mixing time (each 1 min long) and (ii) a data acquisition period of 57 min. The latter consisted of 3 cycles with waiting time before each measurement lasting for 15 min.

Neutrophils were suspended in PBS (glycolysis stress test medium, w/o glucose and pyruvate supplementation, pH 7.4), plated (300,000 cells/well) in 160 μL on Agilent Seahorse 8-well XFp Cell Culture Miniplate and allowed to adhere for 30 min at 37 °C. Just before running the assay, neutrophils were stimulated with LPS (75 µg/mL final well conc.) and immediately placed in the instrument. The assay workflow was as follows: (1) injection of glucose (10 mM; rate of glycolysis), (2) injection of ATP synthase inhibitor - oligomycin (1 µM; cellular maximum glycolytic capacity). (3) Lastly, 2-DG (50 mM) was injected to abolish glycolysis. Measurements after each injection consisted of a mixing time of 3 min each, and a data acquisition period of 18 min consisting of 3 cycles. The difference between glycolytic capacity and glycolysis rate defines glycolytic reserve. Three cycles of baseline measurement (total duration of 18 min) were taken prior to the addition of glucose. ECAR, prior to glucose injection, is referred to as nonglycolytic acidification. All parameters were calculated using Agilent Seahorse XF Glycolysis Stress Test Report Generator.

Neutrophils were suspended in sterile/filtered HBSS(+) w/o sodium bicarbonate (Gibco, Waltham, MA, USA) supplemented with 1 mM sodium pyruvate (Sigma–Aldrich, USA), 2 mM L-Glutamine (Sigma–Aldrich, Saint Louis, MO, USA), 10 mM D-glucose (Lonza Bioscience, Basel, Switzerland, USA), 5 mM HEPES (Sigma-Aldrich, Saint Louis, MO, USA), adjusted to pH 7.4 with 0.1 N NaOH (Sigma–Aldrich, Saint Louis, MO, USA). Cells were plated (300,000 cells/well) in 160 μL on Agilent Seahorse 8-well XFp Cell Culture Miniplate. Just before running the assay, neutrophils were stimulated with LPS (75 µg/mL final well conc.) and immediately placed in the instrument. Following inhibitors required to determine the dependency, capacity, and flexibility of cells for long chain fatty acids oxidation were used: Etomoxir (40 μM, final well conc., port A, 20 µL; Sigma–Aldrich, Saint Louis, MO, USA)—an inhibitor of long chain fatty acid oxidation, UK5099 (20 μM, final well conc., port B, 22 µL, Sigma–Aldrich, Saint Louis, MO, USA)—an inhibitor of the glucose oxidation, BPTES (30 μM, final well conc., port C, 25 µL, Sigma–Aldrich, Saint Louis, MO, USA)—an inhibitor of the glutamine oxidation. Three cycles of baseline measurements (57 min) with a mixing time (1 min) and waiting time before each cycle (15 min) were taken. Subsequently, measurements after each inhibitor were performed. They consisted of a mixing time of 3 min and a data acquisition period of 30 min consisting of 3 cycles. Fatty acid (FA) dependency was tested by first injecting Etomoxir, followed by inhibition of the two alternative pathways (UK5099/BPTES). FA capacity was tested by first injecting UK5099/BPTES, followed by injection of Etomoxir. Fuel Flexibility was calculated as the difference between capacity and dependency. All parameters were calculated using Agilent Seahorse XF Mito Fuel Flex Test Report Generator.