Xf base media

Primary myoblast isolation and confirmation of myoblast phenotype has been previously published (Simon et al. 2014, 2017b). Myoblasts were seeded (60,000 cells/well) in triplicates on a Seahorse culture plate and cultured in Ham's‐F12 media with 10% FBS, 2% l‐Glutamine and 2.5 ng/mL hFGF for 24 h. Cells from passage 3 to passage 4 were used for all experiments described. Media were then changed to XF Assay Medium [XF Base Media (Cat No. 102353‐100, Seahorse Bioscience) with sodium pyruvate, l‐glutamine and glucose) for 1 h before mitochondrial function was assessed by measuring cellular respiration rates with a Seahorse Flux Analyzer XF‐24 (Agilent technologies, Santa Clara, CA) as specified by the manufacturer. Respiratory parameters (basal and maximal oxygen consumption rates (OCR), proton leak, and spare respiratory capacity) were assessed in response to addition of oligomycin (1 μmol/L), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP; 2 μmol/L), and rotenone/antimycin A (0.5 μmol/L). Values were normalized to protein content for each well and represented as normalized values to the controls. Myoblasts isolated from control and CBA/SIV/ART+ macaques were concomitantly cultured with Formoterol (30 nmol/L) for 24 h.

To assess mitochondrial bioenergetics, NSCs were grown in a Seahorse XFe 24 microplate (Seahorse Biosciences/Agilent Technologies, Santa Clara, CA, USA) coated with Poly-L-Ornithine and fibronectin to 80% confluency. NSCs were treated with 0.5–1 μM cisplatin or vehicle for 12 h, washed with serum-free media, and incubated for 1 h at 37 °C in XF base media (Seahorse Biosciences) supplemented with 11 mM glucose, 2 mM glutamine, and 1 mM pyruvate. Oligomycin (2 mM), FCCP (4 mM), and rotenone/antimycin A (2 mM each) were used with a 3-time repeat of a 2-min mix, 3-min wait, and 2-min measure assay cycle. Oxygen consumption rates were normalized to the total protein content of each well. Basal respiration, adenosine triphosphate (ATP)-linked respiration, proton leak, and maximal respiratory capacity were determined as described previously [7 (link), 23 (link)].

Mitochondrial oxygen consumption rates (OCRs) and extracellular acidification rates (ECAR) were measured using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience). Cells were seed at a density of 20,000 cells in XF24 cell culture microplates and were continuously incubated for 12 h in hypoxia. The culture medium was changed to XF base media as recommended by Seahorse Bioscience before measurement. The ATP synthase inhibitor Oligomycin A (2 μM), the ATP synthesis uncoupler carbonyl cyanide-4-trifluoromethoxyphenylhydrazone FCCP (1.5 μM), the complex I inhibitor rotenone (2 μM), and complex III inhibitor antimycin A (2 μM) were used to determine OCR parameters. OCR is shown in pmol/min/10⁵ cells and ECAR is shown in mpH/min/10⁵ cells. All experiments were performed in triplicate.

Oxygen consumption rates (OCRs) were quantified using the XF Extracellular Flux Analyzer (Seahorse Bioscience). α-Syn-N2a cells were seeded at a density of 5.0 × 10⁴ cells/well employing a Seahorse plate coated with 0.2% gelatin and cultured overnight. Cells were equilibrated with XF Base media (Seahorse, catalog number 102353-100) at 37 °C for 1 h in an incubator lacking CO₂. Mitochondrial stress was measured using an Agilent Seahorse XF Cell Mito Stress Test Kit (Seahorse) based on the manufacturer’s protocol followed by sequential treatments with oligomycin (1.4 μM), FCCP (1.0 mM), and rotenone/antimycinA (0.5 μM) (Seahorse).

Glycolytic rates were measured using the XF^e24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA). After siRNA, chemical inhibitors, or flow experiments, HAECs were collected and seeded at a density of 4 × 10⁴/well on Seahorse plates and allowed to adhere for 4 hr in a standard incubator. Cells were next equilibrated with XF Base media (Seahorse) at 37°C for one hour in an incubator lacking CO₂. Glycolysis stress test was performed by sequential treatments with glucose (10 mM), oligomycin (1.0 μM) and 2-DG (100 mM)). Mitochondrial stress test was performed by sequential treatments with oligomycin (1.0 μM), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (1.0 μM), and rotenone/antimycin A (1.0 μM each). For determination of oxidative phosphorylation substrate utilization, cells were equilibrated in XF Base media supplemented with glucose (5 mM) and glutamine (2 mM). For glucose utilization determination, UK5099 (2 $\text{μ}$ M) and then BPTES (3 μM)/Etoxomir (4 μM) (4 $$ M) or BPTES (3 μM)/Etoxomir (4 μM) and then UK5099 (2 μM) were added sequentially. Dichloroacetate (DCA) at 4 mM was maintained in the media at all times for DCA experiment. All chemicals form Sigma-Aldrich.

Metabolic changes were monitored by an extracellular flux analyzer XFe96 (Seahorse Bioscience). Cells were resuspended in XF Base Media (DMEM, Seahorse Bioscience) supplemented with 2 mM glutamine, and seeded in a Seahorse Bioscience 24-well plate. After 3 hr, cells were treated with medium, LPS + IFN-γ or IL-4, and incubated for 24 hr at 37°C in an atmosphere of 5% CO₂. To measure the extracellular acidification rate (ECAR, glycolysis indication), rotenone plus antimycin A (Rot/AA, 1 μM each), and deoxy-2-glucose (2-DG, 80 mM) were added sequentially. For measuring the oxygen consumption rate (OCR, mitochondrial respiration indication), cells were sequentially treated with oligomycin (1 μM), N⁵,N⁶-bis (2-Fluorophenyl)- (1,2,5) oxadiazolo (3,4-b) pyrazine-5,6-diamine (BAM15, 2 mM), to cause mitochondrial uncoupling, and rotenone plus antimycin A (Rot/AA, 1 μM each). All chemicals were purchased from Calbiochem (Burlington, MA, USA).