Seahorse xf assay media

Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation with lympholyte mammal (1.086 gm/cm³) according to manufacturer instructions (Cedarlane). Single-cell suspensions from BAL were prepared by centrifugation at 500×g followed by RBC lysis with Gey’s solution. 2 × 10⁵ or 4 × 10⁵ PBMCs and 1 × 10⁵ BAL cells suspended in Seahorse XF assay media (Agilent Technologies, Inc), were seeded onto CellTak (Corning Inc.) coated 8 or 24 well cell culture microplates, respectively. Cell adherence to microplates was achieved prior to assaying by centrifuging plates at 500×g for 5 min with no brake followed by a 30-min incubation at 37 °C in the absence of CO₂. OCR and ECAR were measured using a Seahorse XFp or Seahorse XFe24 bioanalyzer (Agilent Technologies, Inc). OCR and ECAR were measured basally and following the injection of 1 μM oligomycin, 2 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), 0.5 μM rotenone + antimycin A, and 50 mM 2-deoxyglucose.

To determine the balance of oxidative vs. fermentative energy production in B16F10 cells, OCR and ECAR were measured using a Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). Cells were seeded into XFp cell culture 8-well miniplates at a starting density of 4 × 10⁴ cell/well in duplicate and cultured under standard conditions overnight. Then, the cells were treated with 0, 5, or 10 µM of DEA for 3 h. Prior to the measurement, the medium was replaced with Seahorse XF Assay Media (Agilent, Santa Clara, CA, USA) pH 7.4 supplemented with 10 mM of glucose, 2 mM of L-glutamine, and 1 mM of pyruvate. Mitochondrial stress test was performed using the following inhibitors at the indicated final concentrations: 1 µM of oligomycin, 1 µM of FCCP, and 1 µM of rotenone–antimycin A. In each experiment, two wells without cells were running to assess the non-cellular oxygen consumption, which was subtracted from the corresponding OCR value. The OCR and ECAR data were normalized to the mg protein content using the Micro BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) for the measurement of protein concentrations. No other data correction was applied. These experiments were repeated three times.

Mitochondrial respiration parameters were measured using a Mito Stress Test Kit and XF96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s protocol. In brief, 15,000 cells/well were plated on a 96-well plate on Cell-Tak (Corning) coated wells in their standard growing media and cultured overnight. The next day, cells were treated with drugs and incubated at 37°C, 5% CO₂ for the indicated treatment time. Seahorse XF assay media (Agilent) was used to wash and remove the standard growing media from the cell plate and the sensor plate was prepped with standard Mito Stress Test drugs. After calibration of the sensor plate on the XF96 Extracellular Flux Analyzer, the cell plate was inserted into the machine and each parameter was calculated with 4 measurements separated by 5 min intervals following injections of drugs. Basal respiration measurement was followed by injections of oligomycin (2 µM), FCCP (0.5 µM) and Rotenone/Antimycin (1µM each), respectively. Individual respiratory parameters were then calculated according to the manufacturer’s protocol.

Cells were collected and counted as described above. The diff. THP-1 cells were PMA-differentiated directly on XFp cell culture mini plates. A total of 40,000 cells were placed onto the XFp cell culture mini plates and incubated with EC₂₀ doses of AgNPs for 12 h, together with vehicle controls; each condition was tested in triplicate. Before measurement, the cell culture medium was replaced with Seahorse XF Assay Media (Agilent, Santa Clara, CA, USA) supplemented with 10 mM of glucose, 2 mM of L-glutamine, and 1 mM of pyruvate. Using a Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA), a Seahorse XF Cell Mito Stress Test was performed. The oxygen-consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured. For details of the applied inhibitors, explanations of the Seahorse XF Cell Mito Stress Test principles, and details of protein quantification, please see the Supplementary Material. The OCR and ECAR values were normalized to the protein content. The experiments with each cell type were performed at least three times.

Mitochondrial function was analyzed using a Seahorse XF96 extracellular flux analyzer (Agilent Technologies, Carlsbad, CA, USA). After culturing for 14 days, the hiPSC-CM tissue was dissociated into a single-cell suspension using 0.25% trypsin/EDTA (Thermo Fisher) and then seeded in a microplate (XF96, Agilent Technologies) at a density of 2×10⁴ cells/well. After 3 days of culture, the culture medium was replaced with base medium (Seahorse XF assay media; Agilent Technologies, Carlsbad, CA, USA) supplemented with 1 mM sodium pyruvate. Substrates and inhibitors were added during measurements to attain a final concentration of 3.5 μM 4-(trifluoromethoxy) phenylhydrazone (FCCP; Seahorse Bioscience, Billerica, MA, USA), 1 μM oligomycin, 0.5 μM antimycin, and 0.5 μM rotenone for the MitoStress assay.

Oxygen consumption rate (OCR) was measured using a Seahorse XFe96 extracellular flux analyzer (Agilent, Santa Clara, CA). HBEC3-KT cells (1.5×10⁴ per well) were seeded into a XFe96 microplate and grew overnight in complete media. 1 h before the assay, the media was changed to Seahorse XF assay media (Agilent) and incubated in a non-CO₂ incubator at 37°C. The microplate was loaded onto the analyzer and basal respiration in these cells were recorded by real-time measurement of OCR. Then 25 μl of PBS, ODN and/or mitochondria inhibitors prepared in the assay medium were sequentially injected into each culture well (in 150 μl of assay media) via drug delivery ports. The final working concentrations of these testing regents in culture wells are ODN 1.2 μM, oligomycin 10 nM, and antimycin 1 μM. After injection of each regent, OCR was again measured. At the end of the assay, the number of viable cells was determined using trypan blue. OCR measurements were normalized to final cell numbers. OCR is expressed in pmole min⁻¹. Studies were performed a minimum of 3 times with 12 biological replicates per condition.