Seahorse xf24 cell culture microplate

ECAR and OCR were analysed with Seahorse XFe24 Analyzer (Agilent). Cells were seeded on Seahorse XF24 cell culture microplates (Agilent) at density of 50 × 10³ cells/well. 24 h after seeding, growth medium was replaced with Seahorse XF DMEM medium (Agilent) supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM glutamine. Cells were incubated for 1 h at 37 °C in normal atmosphere (no additional CO₂) and then transferred into Seahorse. In Seahorse, cells were treated with ouabain or vehicle for ~ 3½ h, which was followed by successive treatment with 1 μM oligomycin, 0.75 μM FCCP, and a combination of 1 μM rotenone and 1 μM antimycin A.

Human fASM were plated at a density of 2 × 10⁴ cells/well in a growth medium using Agilent Seahorse XF24 Cell Culture Microplates (Agilent #100777-004, Santa Clara, CA, USA). After 24 h, cells were exposed or treated accordingly. 24 h later, mitochondrial stress tests were performed using a Seahorse XF_e Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Seahorse assay medium consisted of XF Base Medium (Agilent Technologies #103334-100, Santa Clara, CA, USA) supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine at pH 7.4. Seahorse XFe24 FluxPak sensor cartridge was hydrated for 24 h prior to assaying using Seahorse XF Calibrant Solution (Agilent #102340-100, Santa Clara, CA, USA). Stock mitochondrial stress test reagents were prepared using Sigma-Aldrich compounds (oligomycin: Sigma #75351; FCCP: Sigma #c2920; Antimycin A: Sigma #A8674; Rotenone: Sigma #R8875; St. Louis, MO, USA). The final concentration of mitochondrial stress test reagents are as follows: 1 µM oligomycin, 1.25 µM FCCP, 1 µM Antimycin A, and 1 µM Rotenone. Seahorse Bioanalyzer protocol is as follows: 3 cycles per compound of 1’ mix, 2’ wait, 3’ measure. Normalization was done by in-situ cell counting using 1 ug/mL Hoechst 33342 Solution (Thermo Scientific #62249, Waltham, MA, USA) and Cytation 5 imaging (BioTek/Agilent, Winooski, VT, USA).

glucose or glutamine dependency/flexibility was determined using the Seahorse XF Mito Fuel Flex test kit and a Seahorse XFe24 Flux Analyzer (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocols. Briefly, cells were plated at 70–80% confluency in Seahorse XF24 Cell Culture Microplates (Agilent) in triplicates with XF base medium (Agilent) containing 25 mM glucose (Sigma), 2 mM glutamine (Thermo Fisher) and 1 mM sodium pyruvate (Thermo Fisher). Three replicate oxygen consumption rate (OCR) measurements were acquired at baseline and six measurements were taken after the first injection of UK5099 (2 μM) or BPTES (3 μM) and second injections of etomoxir (4 μM) and BPTES or UK5099 for quantification of glucose or glutamine dependency, respectively. The values for glucose or glutamine dependency/flexibility were calculated from each average OCR values as described in the Seahorse user guide.