Agilent seahorse xfp cell mito stress test kit

Metabolic flux was measured using the Seahorse XFp Analyzer (Agilent, Santa Clara, CA) as previously described^{12 (link)}, and according to manufacturer protocols. Cells were plated at a density of 1 × 10⁴/well using Seahorse assay media (Agilent, 103010-100) supplemented with 0.9% (w/v) glucose, 1 mM Sodium Pyruvate, and 2 mM L-Glutamine. The Agilent Seahorse XFp Cell Mito Stress Test Kit was used to acquire serial measurements of oxygen concentrations before, and following administration of 1 μM oligomycin, 1 μM FCCP, and 1 μM Rotenone and Antimycin cocktail. To determine mitochondrial fuel dependency for each of the cell lines, the Agilent Seahorse Mito Fuel Flex Test (Agilent, 103270-100) was used. Specifically, the test uses 1 μM UK5099, BPTES, or Etomoxir to inhibit mitochondrial substrate transport for glucose, glutamine, and fatty acids, respectively. This allowed determination of relative dependency on each individual pathway through analysis of oxygen consumption rates during concomitant inhibition of the two alternative fuel sources. The oxygen consumption from individual substrates was calculated as a ratio relative to total basal fuel metabolism (100%) and displayed as a proportion of total oxygen utilization for each substrate.

The Seahorse XFp mitochondrial stress test was performed using an Agilent Seahorse XFp Cell Mito Stress Test Kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer's instructions. Briefly, hVSMCs incubated with normal or CKD serum were seeded at a density of 1 × 10⁴ cells per well on XFp microplates. Then, the cells were rinsed once and XF assay medium was added. After three measurements of baseline OCR, respiratory chain inhibitors including 1 µM Oligo, 1 µM FCCP and 0.5 µM Rot/Ant were sequentially injected into each well. Three OCR readings were taken after the addition of each inhibitor and before the automated injection of the subsequent inhibitor. OCR was automatically calculated by Wave software version 2.4.0 (Agilent Technologies) and normalized by cell numbers.

8.000 fibroblasts/well were seeded in Seahorse XFp cell culture miniplates 24 hours before measurements. Analyses of respiration and inhibition/uncoupling of respiratory chain complexes were done using Agilent Seahorse XFp Cell mito stress test kit (Agilent Technologies, Santa Clara, CA, USA) in a Seahorse XFp System (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer’s instructions.