Seahorse xfp cell mito stress test

Seahorse XFp Cell Mito stress test (Cat# 103010-100, Agilent Technologies, Santa Clara, CA) was performed according to the manufacturer’s protocol using an XFp instrument. CD4 T cells from healthy subjects were purified from PBMCs, cultured in complete RPMI-1640 medium with 10% FBS, and treated with 5 μM CPT or DMSO for 2 days. One day prior to the assay, Seahorse mini cartridges were hydrated overnight in a non-CO₂ incubator. On the day of the assay, the treated cells were seeded onto mini culture plates pre-coated for 1 h with poly-D-lysine (Thermo Fisher Scientific). Approximately 100,000 cells per well were cultured in Seahorse XF RPMI assay medium supplemented with 1.0 mM of glucose, 100 µM of pyruvate, and 1.0 mM of glutamine. The following inhibitors from the Cell Mito stress test kit were added to the culture media in this order: 2.0 μM of Oligomycin, 1.5 μM of FCCP, and 2.0 μM of Rotenone/Antimycin A, and the related three sequential measurements were recorded. Data analysis was performed using the Seahorse Wave software and the Seahorse Mito stress test report generator.

Seahorse XFp analyzer (XFp, Seahorse Biosciences, MA) was used to evaluate the mitochondria oxygen consumption rate (OCR) of C2C12 myotubes. After C2C12 myoblasts were seeded and differentiated in Seahorse XFp miniplates, myotubes were pretreated with AN07 (0.1 or 1 μM) for 1 h followed by the addition of LPS (100 ng/mL) for 24 h. Before assay, the fluorescence probes were activated by Agilent Seahorse XF calibrant at 37 °C in a non-CO2 incubator overnight. Subsequently, the culture medium was replaced with Agilent Seahorse XF base medium (with 1 mM pyruvate, 2 mM glutamine, 10 mM glucose, and pH adjusted to 7.4), and the miniplates were then placed in a non-CO₂ incubator at 37 °C for 1 h. The key parameters of mitochondrial function were measured using Seahorse XFp Cell Mito Stress Test, which were obtained at the baseline and following sequential injection of oligomycin (1 μM), FCCP (1 μM), and a mixture of antimycin A plus rotenone (AA/ROT, 1 μM).

Seahorse XFp Cell Mito Stress Tests (Cat# 103010-100; Seahorse, Agilent Technologies) were completed according to the manufacturer’s protocol using an XFp instrument. Briefly, CD4 T cells were purified from PBMCs, cultured in 10% FBS cRPMI with 30 IU/ml IL-2 (Cat #589104; BioLegend), and stimulated by 1μg/ml anti-CD3 (Cat # 300333) and 2 μg/ml anti-CD28 antibodies (Cat # 302943; BioLegend) for 3 days. One day before the assay, seahorse mini-cartridges were hydrated overnight in a non-CO₂ incubator. On the day of the assay, seahorse mini plates were coated with 25 μl of 0.1 mg/ml poly-D-lysine (Cat #A3890401; ThermoFisher Scientific) for 1 h. Stimulated CD4 T cells were washed by DPBS and then plated onto pre-coated plates (2 x 10⁵/well) with Seahorse XF RPMI-1640 medium with 1.0 mM Glucose, 100 µM Pyruvate and 1.0 mM Glutamine. Data was analyzed using the Seahorse Wave software.