12 well culture plate

Aliquots of 0.5 × 10⁵ cells were seeded into a 12-well culture plate (IWAKI). For ATP reduction, the cells were incubated on coverslips with 10 mM sodium azide and 50 mM 2-deoxyglucose in HBSS (Gibco) for 10 min. To measure ATP, Cell ATP Assay Reagent (300-15363, Toyo B-Net Co. Ltd.) was used according to the manufacturer’s instructions. Bioluminescence was measured using a Lumat LB 9507 tube luminometer (EG&G Berthold). A standard plot of ATP concentration versus bioluminescence intensity verified that our measured ATP concentrations fell within a linear range. Both the reaction and measurement were performed at 23°C in the dark. The incubation time was 5 min from the addition of assay reagent to measurement.

For ATP reduction, cells were incubated with 10 mM sodium azide and 50 mM 2-deoxyglucose in DMEM for 40 min at 37°C. To measure ATP, aliquots of 0.5 × 10⁵ cells were seeded into a 12-well culture plate (IWAKI) and Cell ATP Assay Reagent (300-15363, Toyo B-Net Co. Ltd.) was used according to the manufacturer’s instructions. Bioluminescence was measured using a Lumat LB 9507 tube luminometer (EG&G Berthold). A standard plot of ATP concentration versus bioluminescence intensity verified that our measured ATP concentrations fell within a linear range. Both the reaction and measurement were performed at 23°C in the dark. The incubation time was 5 min from the addition of assay reagent to measurement.
Magnesium Green AM (K_d ∼ 1.0 mM; M3735, Thermo Fisher Scientific) was applied to the DMEM culture medium at 10 μg/ml with 0.02% Pluronic F-127 (P3000MP, Thermo Fisher Scientific), and the cells were incubated at 37°C for 1 hour. Magnesium Green fluorescence was measured by DeltaVision equipped with an Olympus PlanApoN 60× objective (NA 1.42) and an sCMOS camera with a FITC filter by using Softworx software. Cell culture conditions (37°C, 5% CO₂, and humidity) were maintained in a live-cell chamber under the microscope. The nucleoplasm intensity was measured using Softworx software and plotted after subtracting background signals outside the cells.