Atp bioluminescence assay kit hs 2

ATP production rate was measured in digitonin-permeabilized RPTC (0.01%
final concentration) respiring at state 3 using the method of Borkan et al. (1993) (link), modified as described
previously (Nowak et al., 2006 (link)). The
assay was carried out using 5 mM glutamate + 5 mM malate or 10 mM succinate +
0.1 μM rotenone as the energizing substrates. The reaction was terminated
by adding an aliquot of ice-cold perchloric acid (3% final concentration) and
the suspension was spun down (15,000×g for 1 min). The supernatant was
neutralized to pH 7.5 and analyzed for ATP content using the luciferase method
and ATP Bioluminescence Assay Kit HS II (Roche, Mannheim, Germany).
Intracellular ATP content was measured in freshly prepared RPTC lysates
using the luciferase method and ATP Bioluminescence Assay Kit HS II as described
previously (Nowak et al., 2004 (link)).

For all wing posture assays, male flies at 7-day and 14–15-day old and aged at 29°C were used. 20 male flies were collected/raised in one vial and 3 independent vials were counted per genotype. Measurements of ATP contents in thoracic muscle were performed as described previously (Gehrke et al., 2015 (link)), using a luciferase-based bioluminescence assay (ATP Bioluminescence Assay Kit HS II, Roche Applied Science). For each test, three thoraces were dissected by removing heads, wings, legs and abdomen from whole flies, and the remaining part quickly homogenized in 100 μl lysis buffer. The tissue lysates were then boiled for 5 min at 100°C and briefly cleared by centrifugation at 20,000 g for 2 mins. The supernatant was transferred to a new tube and kept on ice. 2.5 μl of cleared tissue lysate was-mixed with 187.5 μl dilution buffer and 10 μl luciferase reagent.The luminescence signal wasimmediately measured by a Lumat LB 9507 tube luminometer (Berthold Technologies). For each genotype or drug treatment group, at least 3~4 independent tests were assayed.