Bioluminescence analysis of ATP in HBSS and Tyrode’s buffer

ATP was serially diluted into 1 to 0.031 μM with HBSS (138 mM NaCl, 5.33 mM KCl, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 5.56 mM glucose, 0.41 mM MgSO₄, 0.49 mM MgCl₂ and 1.26 mM CaCl₂, pH 7.3) or modified Tyrode’s buffer (137 mM NaCl, 2.9 mM KCl, 0.34 mM Na₂HPO₄, 12 mM NaHCO₃, 5 mM HEPES, 5 mM glucose, 1 mM MgCl₂ and 1 mM CaCl₂, pH 7.3) and added into 96-well plate (100 μl). In the presence or absence of CoA (30 μM), enzyme work solution (2 ×) containing DTT (2 mM), luciferin (1 mM) and luciferase (2.5 μg/ml) was also prepared with HBSS or Tyrode’s buffer, and added into 96-well plate (100 μl) to initiate bioluminescence reaction, then RLU was analyzed at different time points.