Seahorse mito stress test generator

Real-time mitochondrial function in AML cells and healthy PBMCs was assessed using the Seahorse XF Cell Mito Stress Test kit (Agilent Technologies, Santa Clara, CA, USA) on Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Briefly, cells, untreated or treated for 2 h with selected drug combinations at the 1/1,000 dose of maximal selectivity, were plated on a Cell-Tak (ThermoFisher) coated XF96 96-well microplate using XF base media, supplemented with 1 mM pyruvate, 2 mM L-glutamine, 5 mM glucose. OCR measurements were recorded after port injection starting with oligomycin (1.5 μM) followed by FCCP (1 μM), and lastly, a combination of antimycin A and rotenone (0.5 μM). All measurements were normalized to the number of viable cells. Basal respiration, ATP-coupled respiration, maximal and spare capacities, ECAR values, as well as non-mitochondrial respiration were recorded per each condition. The results were analyzed in Seahorse Mito Stress Test Generator (Agilent Technologies).

Real-time mitochondrial function in AML cell lines, healthy PBMCs, and mitocan-resistant SKOV3 cells was assessed using the Seahorse XF Cell Mito Stress Test kit (Agilent Technologies, Santa Clara, CA, USA) on Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies). Briefly, cells, untreated or treated with LD50 of CCCP, DOX, 2-DG, or the combination of CCCP and 2-DG (LD25), were plated on a Cell-Tak (ThermoFisher)-coated XF96 96-well microplate using XF base media supplemented with 1 mM pyruvate, 2 mM l-glutamine, 5 mM glucose. OCR measurements were recorded after port injection starting with oligomycin (1.5 µM) followed by FCCP (1 µM), and lastly, a combination of antimycin A and rotenone (0.5 µM). All measurements were normalized to the number of viable cells. Basal respiration, ATP-coupled respiration, maximal and reserve capacities, ECAR values, as well as nonmitochondrial respiration were recorded per each condition. The results were analyzed in a Seahorse Mito Stress Test Generator (Agilent Technology).