Atp assay reaction mixture

Cells grown in 24-well plates were lysed in PBS buffer containing 0.5% phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and boiled at 95°C for 10 min. Following centrifugation at 10,000 g for 5 min to remove cell debris, protein content in the supernatant was measured and samples were adjusted to have equal concentrations. Samples were then diluted 1:10 in PBS/0.5% phosphatase inhibitor buffer. For the detection of ATP levels, a commercially available ATP assay reaction mixture (Sigma-Aldrich) containing luciferin and luciferase was used (50 μl of adjusted sample plus 100 μl of assay mix was added to a black 96-well plate). Standards were prepared by serial dilutions of ATP disodium salt hydrate (Sigma-Aldrich) to obtain concentrations ranging from 1000 to 7.8 nM.

To determine intracellular ATP, cells grown in 24-well plates were scratched and sonicated in PBS buffer and boiled at 95°C for 10 min followed by centrifugation at 10 000 × g for 5 min for the removal of cell debris. For the detection of ATP levels, a commercially available ATP assay reaction mixture (Sigma) containing luciferin and luciferase was used. To a black 96-well plate, 50 μl of sample and 100 μl of assay mix were added. Standards were prepared by serial dilutions of ATP disodium salt hydrate (Sigma) to obtain final concentrations ranging from 1000 to 7.8 nM.