Seahorse xf extracellular ux analyzer xfe96

The OCR assays were performed using the Seahorse XF extracellular ux analyzer (XFe96) (Agilent Technologies, Santa Clara, USA) with the Seahorse XF Cell Mito Stress Test Kit (103708-100, Seahorse) and Seahorse XFe96 FluxPak Plates (102416-100, Seahorse) according to the manufacturer's instructions. Brie y, 6000-8000 cells per well were seeded into the Seahorse XFp cell culture microplate overnight or treated with TAT-Pep for another 24 h. The probe plate was hydrated with deionized water for at least 4 h and then changed to calibration solution for another 1 h at 37 °C in a cell incubator without CO 2 before the assays run. The base medium (pH=7.4) was supplemented with D-glucose (10 mM), Pyruvate (1 mM) and L-glutamine (2 mM) to make the detection solution. Next, cells were washed using the detection solution and incubated in a cell incubator without CO 2 before the assays run. After baseline measurements, ATP synthase inhibitor Oligomycin (1 μM), the OXPHOS uncoupling agent FCCP (0.5 or 1 μM) and Respiratory Chain inhibitor Rotenone/Antimycin A (0.5 μM/0.5 μM) were sequentially injected into each well at the indicated time points. Data was recorded and analyzed by the Seahorse XF Wave software. Maximal Respiration was calculated as the difference between the average value of time point 7,8,9 and that of time point 10,11,12.

The ECAR assays were performed using the Seahorse XF extracellular ux analyzer (XFe96) (Agilent Technologies, Santa Clara, USA) with the Seahorse XF Cell Glycolysis Stress Test Kit (103710-100, Seahorse) and Seahorse XFe96 FluxPak Plates (102416-100, Seahorse) according to the manufacturer's instructions. Brie y, 6000-8000 cells per well were seeded into the Seahorse XFp cell culture microplate overnight or treated with TAT-Pep for another 24 h. The probe plate was hydrated with deionized water for at least 4 h and then changed to calibration solution for another 1 h at 37 °C in a cell incubator without CO 2 before the assays run. The base medium (pH=7.4) was supplemented with L-glutamine (2 mM) to make the detection solution. Next, cells were washed using the detection solution and incubated in a cell incubator without CO 2 before the assays run. After baseline measurements, D-glucose (10 mM), ATP synthase inhibitor Oligomycin (1 μM), the glycolysis inhibitor 2-DG (50mM) were sequentially injected into each well at the indicated time points. Data was recorded and analyzed by the Seahorse XF Wave software. Glycolytic capacity was calculated as the difference between the average value of time point 7,8,9 and that of time point 10,11,12.