2.13. ATP/ADP luciferase assay

ATP and ADP concentrations were measured using a Luciferase based assay as described by Strehler et al. [29] and performed by Martin et al. [30]. In brief, approximately 10 mg of clamp frozen tissue was weighed out on dry ice into pre-cooled Precellys tubes (Hard tissue homogenising CK28-R - 2 mL; Bertin Instruments, France). After weighing, 100 μL/mg of ice-cold perchloric acid extractant (3% (v/v) HClO₄, 2 mM Na₂-EDTA, 0.5% Triton X-100) was added to each sample. Samples were then homogenised using a Precellys 24 tissue homogeniser (6500 rpm, 2 × 15 s, Bertin Instruments, France) transferring samples back onto wet ice between cycles. Following homogenisation, samples were centrifuged (10,000g, 1 min, 4 °C) and the supernatant diluted in ice cold perchloric acid extractant to a concentration of 1 mg frozen tissue per mL. Samples along with ATP and ADP standards were neutralised to pH 7 using potassium hydroxide solution (2 M KOH, 2 mM Na₂-EDTA, 50 mM MOPS). To measure ADP concentration, 250 μl of neutralised sample was added to 250 μL ATP sulfurylase assay buffer (20 mM Na₂MoO₄, 5 mM GMP, 0.2 U ATP sulfurylase (New England Biolabs, USA), in Tris-HCl buffer (100 mM Tris-HCl, 10 mM MgCl₂ (pH 8.0)) and incubated for 30 min at 30 °C to convert the sample's ATP to AMP. ADP samples were then heated at 100 °C for 5 min and then cooled on ice. Standards (100 μL), samples for ATP measurement (100 μL) or samples for ADP measurement (200 μL) (in duplicate) were then added to 400 μL Tris-acetate (TA) buffer (100 mM Tris, 2 mM Na₂-EDTA, 50 mM MgCl₂, pH 7.75 with glacial acetic acid) in luminometer tubes. To measure ADP concentration, 10 μL pyruvate kinase solution (100 mM PEP, 6 U pyruvate kinase suspension (Sigma #P1506)) was added to one set of samples for ADP measurement and incubated for 30 min at 25 °C in the dark to convert ADP to ATP. The other duplicate tube (without addition of pyruvate kinase solution) served as an ADP ‘blank’ value. The samples were then all assayed for ATP content in a Berthold AutoLumat Plus luminometer by addition of (100 μL) Luciferase/Luciferin Solution (7.5 mM DTT, 0.4 mg/mLBSA, (1.92 μg) luciferase/mL (SIGMA #L9506), 120 μM D-luciferin (SIGMA #L9504), made in TA buffer (25% (v/v) glycerol)), delivered via auto injection, protected from light. Bioluminescence of the ATP- dependent luciferase activity was measured for 45 s post injection and the data quantified against standard curves.