Oxygen consumption was measured using a Seahorse XFe96 Analyzer (Agilent) at 20 °C similar to that described in an earlier study39 (link). 15 worms on Day 3 of adulthood, treated with or without chlorpropamide (100 μmol/L) as described in lifespan assay, were transferred into each well of a 96-well microplate containing 200 μL M9 buffer with 6 wells per group. Basal respiration was measured for a total of 90 min, at 9-min intervals that included a 3-min mix, a 3-min time delay, and a 3-min measurement.
Oxygen consumption of HL-7702 cells was measured using a Seahorse XFe96 Analyzer (Agilent) at 37 °C40 (link). Cells were cultured in 100 mm dishes with DMSO/chlorpropamide (200 μmol/L) for two days and seeded in XF96 V3 Cell Culture Microplates (Agilent) at 10,000 cells/well over night before the experiment. Succinate (10 mmol/L) with rotenone (2 μmol/L) was added to MAS with 0.2% (w/v) BSA as substrates. ADP (final concentration, 4 mmol/L), oligomycin (final concentration, 2.5 μg/mL), FCCP (final concentration, 1 μmol/L) and antimycin A (final concentration, 4 μmol/L) was added into different ports of Seahorse cartage. For permeabilization of cells, 0.001% digitonin was used. Each experimental group was analyzed using three or four replicates.
Oxygen consumption of HL-7702 cells was measured using a Seahorse XFe96 Analyzer (Agilent) at 37 °C40 (link). Cells were cultured in 100 mm dishes with DMSO/chlorpropamide (200 μmol/L) for two days and seeded in XF96 V3 Cell Culture Microplates (Agilent) at 10,000 cells/well over night before the experiment. Succinate (10 mmol/L) with rotenone (2 μmol/L) was added to MAS with 0.2% (w/v) BSA as substrates. ADP (final concentration, 4 mmol/L), oligomycin (final concentration, 2.5 μg/mL), FCCP (final concentration, 1 μmol/L) and antimycin A (final concentration, 4 μmol/L) was added into different ports of Seahorse cartage. For permeabilization of cells, 0.001% digitonin was used. Each experimental group was analyzed using three or four replicates.