Xfp cell mito stress test kit

Oxygen consumption rate (OCR) was measured in freshly microdissected TAL tubules using the XFp Extracellular Flux Analyzer and the XFp Cell Mito Stress Test kit (Seahorse Bioscience, North Billerica, MA). TAL microdissection was performed as described above with following modifications: TAL’s were collected in XF-Base Medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 10 mM glucose (pH 7.4) and seeded in the Seahorse XFp Cell Culture Miniplate containing the same medium and kept for 1 h at 37 °C in ambient air. OCR was measured at basal condition and following the injection of bumetanide (Sigma-Aldrich) at a final concentration of 100 µM. After the measurement, tubules were fixed in 4% PFA and tubule length was assessed using ImageJ software^{35 (link)}.

Primary endothelial cells were seeded in Poly‐d‐lysine‐coated XF96 cell culture microplates (Agilent Technologies, 101085‐004) at 4 × 10⁴ cells/well density in 80 µL growth media. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using XFp extracellular flux analyzers (EFA) (Seahorse Bioscience) and XFp Cell Mito Stress Test Kit. During the assay, cells were exposed to compounds in the following order: 1.0 × 10⁻³m oligomycin, 1.0 × 10⁻³m FCCP, 0.5 × 10⁻³m rotenone/antimycin A.

Analysis of macrophage mitochondrial oxygen consumption rates (OCRs) was performed under different treatments in 8-well plates by using a XFp Cell Mito Stress Test Kit (#103010-100, Seahorse) on an XFp Flux Analyzer. In brief, native BMDMs were seeded into XFp cell culture microplates (#103022-100, Seahorse) and subjected to different treatments. Then, cells were transferred to a CO₂-free incubator and maintained at 37 °C for 1 h after adding a final volume of 175 μl buffer-free assay medium to each well. Following instrument calibration, the cells were transferred to the XFp Analyzer to record cellular oxygen consumption rates. Basal cellular OCRs were recorded without any inhibitors and uncouplers. For the mitochondrial stress test, ATP synthase was inhibited by adding 1 μM oligomycin, followed by 2 μM FCCP-induced mitochondrial uncoupling to achieve maximal OCRs. Finally, nonmitochondrial respiration was determined after injection of a rotenone/antimycin A (1 μM each) combination. Once the XF experiment was completed, cell proteins were extracted to determine the protein concentration for normalization and the OCR was displayed as pmol min⁻¹ μg⁻¹ protein.

The cell OXPHOS and cell glycolysis levels of HepG2 and A549 with or without PPP1R14B-AS1 knockdown were measured using a Seahorse XFp analyzer with a Seahorse XFp Cell Mito Stress Test Kit and a Glycolysis Stress Test Kit in accordance with the manufacturers’ instructions.

Mitochondrial OCRs were analyzed using XFp analyzer^{73 (link)} (Seahorse Bioscience, Waldbronn, Germany) according to the manufacturer's instructions in RPMI medium without glucose and sodium bicarbonate (R1383, Sigma), supplemented with 10 mM glucose (G8644, Sigma) and 1 mM pyruvate (P5280, Sigma) using 2 μM oligomycin, 0.6 μM rotenone/antimycin and 0.7 μM FCCP (XFp cell mito stress test kit, Seahorse Bioscience). In all, 5 × 10⁴ TSCs were seeded 24 h before the beginning of the assay. Data were normalized to protein content after the assay.
Mitochondrial membrane potential (JC1 assay) was assessed using MitoProbe JC-1 Assay Kit for Flow Cytometry (Thermo Fisher Scientific) following the manufacturer's instructions. Briefly, TSCs were cultured in the presence of the mitoprobe 2 μM 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Background measurements were obtained using 50 μM CCCP (carbonyl cyanide 3-chlorophenylhydrazone). JC-1 fluorescent ratio from 590/529 nm (Fl2/FL1 channels) was calculated to estimate the relative membrane potential before and after the indicated treatment.

The OCR and ECAR were measured in XFp extracellular flux analyzers (EFAs) (Seahorse Bioscience) using a XFp Cell Mito Stress Test Kit and a XFp Glycolysis Stress Test kit, respectively. The following parameters were used in the assays: seed cells 8 × 10⁴ per well, Oligomycin 1.0 μM, FCCP 1.0 μM, Rotenone/antimycin A 0.5 μM, glucose 10 mM, and 2-DG 50 mM as indicated.