Intracellular ATP levels determination

Intracellular ATP levels were measured using a firefly luciferase-based bioluminescence ATP Assay Kit (Beyotime Biotechnology). Cell suspensions prepared from 1 plate was diluted to an OD₆₀₀ of 0.2 and centrifuged at 12,000 rpm for 5 min at 4 °C. The obtained cell pellets were used to prepare intracellular ATP solution according to the supplier’s instructions. Cells were mixed with lysis solution and boiled for 2 min to release ATP and to inactivate endogenous ATPase [35, 36]. Then, 20 μL ATP extraction and 100 μL ATP detection solutions were mixed to prepare the luminescent reaction solution.

Luminance (RLU) was recorded using Hidex plate Chameleon™ V. (Finland) and linearly related to the ATP concentration.