2.4. Seahorse Bioanalyzer Mitochondrial Stress Test

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).