Luciferin luciferase assay

The amount of ATP released from whole trigeminal ganglia of mice one week after saline or CFA injection was determined as previously described9 (link) using the luciferin/luciferase assay (Molecular Probes) and a Turner luminometer. Trigeminal ganglia were excised and immediately placed into ice cold (0-4 °C) HEPES buffered, air bubbled artificial cerebrospinal fluid (ACSF: 145 mM NaCl, 2.5 mM KCl, 3.1 mM CaCl₂, 1.3 mM MgCl₂, 10 mM glucose, 10 mM Hepes, pH 7.2). Dissected ganglia were then placed in MatTeck dishes containing 200 μl ACSF, at 37 °C 45 min and aliquots of ACSF solution collected after incubation period and stored at −20 °C until use. The concentration of ATP released by trigeminal ganglia into the ACSF was obtained from standard curves and values normalized to the total amount of protein. Total protein concentration was determined from trigeminal ganglion homogenates (lysis buffer: 150 mM NaCl; 10 mM Tris-base; 1% TritonX-100; pH 7.4) using the BCA reagents (Thermo Fisher).

Cell culture supernatants were measured using a luciferin/luciferase assay (Molecular Probes, Leiden, NL) according to the manufacturer's instruction [48 (link)]. To degrade extracellular ATP in astrocytes cultures, cells were incubated for 1 h with the ATP hydrolyzing enzyme Apyrase (30U/ml, Sigma Aldrich).