Cell mitochondrial stress test

The Seahorse Extracellular Flux XF96 Analyzer (Agilent) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in two assays, Cell Mitochondrial Stress Test (Agilent) and Glycolysis Stress Test (Agilent), following manufacturer instructions. Specifically, 2 × 10⁴ KMRC2 or 786O cells per well were plated in quadruplicates in a 96-well XF culture plate in DMEM XF assay media (Agilent) supplemented with proper concentrations of glucose, sodium pyruvate and L-glutamine. For Mitochondrial Stress Test, oligomycin at 1.5 µM, FCCP at 1 µM and Rotenone/Antimycin A at 0.5 µM were optimized concentrations. For Glycolysis Stress Test, saturating glucose solution at 25 mM, oligomycin at 1 µM and 2-deoxy-glucose (2-DG) at 50 mM were optimized concentrations. After the assay, OCR and ECAR readings were normalized by crystal violet absorbance readings, which are a proxy for total cell content in each well. Cells were fixed and stained with 0.2% crystal violet/25% methanol solution for 10 min at room temperature. Excess stains were washed with PBS and ddH₂O. SDS (1%) was added to solubilize crystal violet stain, following by shaking the plate for 5 min at 350 rpm on MixMate (Eppendorf). Finally, the absorbance was read at 570 nm subtracting the background absorbance at 690 nm.

Total genomic DNA was isolated with the Roche High Pure PCR Template Preparation Kit per manufacturers’ protocol. Mitochondrial DNA copy number was determined by real-time quantitative PCR (Roche LightCycler® 480) as previously described [40 (link)], using LightCycler® 480 SYBR Green I Master (Roche) and 15 ng of DNA per reaction. Oligonucleotide primers for mouse Mito are as follows: (F 5′-CTAGAAACCCCGAAACCAAA and R 5′-CCAGCTATCACCAAGCTCGT and mouse B2M (F 5′-ATGGGAAGCCCGAACATACTG and R 5′-CAGTCTCAGTGGGGGTGAAT (Integrated DNA Technologies).
The mitochondrial membrane potential was determined using JC-1 dye (Cayman) following the manufacturer’s instructions. J-aggregate (excitation/emission = 535/595 nm) and monomer (excitation/emission = 484/535 nm) fluorescence were measured with a SpectraMax M3 (Molecular Devices).
The mitochondrial function was measured using a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies). Cells were cultured in the experimental 25- and 0-nM folate conditions for 4 doublings and then seeded in the same medium and allowed to adhere for 24 h. Basal respiration, ATP production, and extracellular acidification rate were determined following the manufacturer’s instructions for the Cell Mitochondrial Stress Test (Agilent Technologies) and normalized to total cell count.