Dmem xf assay media

Freshly isolated aCMs were plated in 24-well Seahorse XFe™ (Agilent Technologies Inc, Santa Clara, CA, USA) plates and cultured as described above for timepoint analysis. Mitochondrial respiration was assessed 0, 1, 3, and 7 days post-isolation in a Seahorse XFe24 bioanalyzer. Cells were incubated in DMEM XF assay media (#102353-100, Agilent) supplemented with 5 mmol L⁻¹ glucose, 1 mmol L⁻¹ pyruvate, and 2 mmol L⁻¹ glutamine at 37 °C in a CO₂-free incubator for 1 h prior to assay. Injector ports were loaded to provide final concentrations of 1 µmol L⁻¹ oligomycin, 0.5 µmol L⁻¹ FCCP, and 1 µmol L⁻¹ rotenone and 2 µmol L⁻¹ antimycin-A together, respectively according to the mitochondrial stress test protocol provided by Agilent. Maximal respiration was calculated and normalized to total protein concentration as measured by a Bradford assay. Optimal oligomycin and FCCP concentrations were determined by titration.

UM-Chor1 negative controls (NT, LacZ) and PLA2R1 KD (sg1 and sg2) cells were plated on 96-well seahorse plate and were grown at 37 °C in 5% O₂ to reach > 90% confluency. The next day, media was changed to DMEM XF assay media (Agilent) and the plate was allowed to equilibrate for 1 h in the incubator. Cartridges were prepared according to manufactures recommendations. Mitochondrial stress compounds used included Oligomycin (2 μM), FCCP (1 μM; Sigma, C2920) and antimycin A (1 μM). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by XF Seahorse Analyzer (Agilent Technologies).

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.