Seahorse xf cell mitochondrial stress test kit

A Seahorse XF Cell Mitochondrial Stress Test kit (103015-100, Agilent, USA) was used. In brief, primary mouse cardiomyocytes were seeded at a density of 1 × 10⁵/ml in Agilent Seahorse XF24 cell culture microplate (V7-PS, 100777-004, Agilent, USA) on an XF24 extracellular flux analyzer (Agilent Seahorse Bioscience, USA). The Seahorse XF Cell Mitochondrial Stress Test kit was used to provide inhitiors in sequence on plates containing culture medium, including oligomycin (1 μmol/L), fluoro-carbonyl cyanide phenylhydrazone (FCCP; 0.5 μmol/L), rotenone (1 μmol/L), and antimycin A (1 μmol/L). Wave software (Agilent Seahorse Bioscience, USA) was employed for data collection and calculated basic and maximum OCR and other parameters [26 (link)].

Mitochondrial stress detection was conducted by using the Seahorse XF Cell Mitochondrial Stress Test Kit (#103,015–100, Agilent, USA). In brief, the test solution based on DMEM medium (#103,575–100, Agilent, USA) was heated in 37 °C and working solution was prepared for use. Oligomycin, 0.5 µM FCCP, and rotenone/antimycin A were properly prepared into a working solution and added to the dole on the probe plate. Cell culture microplates were removed from a 37 °C CO₂ incubator, and the cells were examined under a microscope to confirm the 90% of confluence. Remove the test solution from the water bath. The cell growth medium in the cell culture microplates was replaced with preheated detection solution using a multichannel pipette, and the cell culture microplates were placed in a CO₂-free incubator at 37 °C for 1 h. Then, run the Seahorse XF 24 (Agilent, USA) on the computer and analyze the data.

The cell oxygen consumption rate (OCR) was measured using an Agilent Seahorse XF Cell Mitochondrial Stress Test kit (103015-100) to characterize the key parameters of mitochondrial function. HK-2 cells in the culture plate were treated with 10 μg/mL of NMs and cultured for 24 h. Subsequently, the user guide for the experiment was followed. Using a Seahorse XFe/XF analyzer (Agilent, Santa Clara, CA, USA), the data were analyzed using the Seahorse XF Mitochondrial Stress Test Report Generator. At least three biological replicates were examined for each experiment.

Rat cardiomyocyte H9c2 cells were purchased from Cell Bioscience Inc. (Shanghai, China). Nuanxin capsule is composed of Red Ginseng, Radix Aconiti Lateralis Preparata, Poria, and Coix seed and was purchased from Guangdong Provincial Hospital of Chinese Medicine (production batch number: 190501, each capsule weighs 0.5 g). Dulbecco's Modified Eagle Medium (DMEM, USA), fetal bovine serum (FBS, Australia), 0.25% Trypsin-EDTA Solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cat. No. #M5655), and phosphate-buffered saline (PBS, USA) were purchased from Gibco (Grand Island, NY, United States), and Annexin V-FITC Apoptosis Detection Kit (No. C1062L) and mitochondrial membrane potential assay kit with JC-1 (JC-1, No. C2006) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Seahorse XF Cell Mitochondrial Stress Test Kit (103010-100) was purchased from Agilent (United States).

The extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) were measured using the Seahorse XFe⁹⁶ Extracellular Flux Analyzer (Seahorse Bioscience). The experiment was performed according to the manufacturer’s instructions. ECAR and OCR were measured using Seahorse XF glycolysis stress test kit and Seahorse XF cell mitochondrial stress test kit (Agilent Technologies). Briefly, cells were transfected or infected as in glucose uptake assay. The transfected or infected cells were harvested, and the cell number was counted. Then, 10,000 cells per well were seeded in the Seahorse XF⁹⁶ cell culture microtiter plate for 10 h. The number of cells in each group was similar. These cells were used to measure ECAR and OCR. After the baseline measurement, for ECAR, glucose, oxidative phosphorylation inhibitor oligomycin, and glycolysis inhibitor 2-DG were sequentially injected into each well at a specified time point. For OCR, oligomycin, the reversible inhibitor of oxidative phosphorylation FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone), and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rote/AA) were sequentially injected. The data were analyzed by Seahorse XFe⁹⁶ Wave software. The results were normalized to the number of cells.