Enliten atp assay system kit

Cells were incubated for 15 min at 37°C with HEPES buffer (pH 7.4) containing AOPCP, a selective inhibitor of ecto-5′-nucleotidase. The buffer was then replaced with fresh HEPES buffer containing AOPCP and incubated in 2% O₂ at 37°C for 5 min. Supernatants were collected at specific time points, and ATP release was measured using the ENLITEN ATP assay system kit (Promega). ATP levels were calculated based on an ATP standard curve.

To measure ATP levels in Drosophila brain samples, the brains of 7-day-old flies were dissected. The ten brain samples were combined, and ATP levels were measured using an ENLITEN ATP assay system kit (Promega, Mannheim, Germany) according to the manufacturer’s instructions and a LuBi microplate luminometer (Micro Digital Ltd., Seoul, Korea). The relative ATP concentration was calculated by dividing the measured ATP concentration by the total protein concentration. The protein concentration was determined by a Bradford assay (Bio-Rad, California, USA). The experiments were independently repeated four times, and the results are presented as the mean ± SD.
To measure ATP levels in the substantia nigra (SN) of mice, mitochondria were isolated from the mouse SN according to a previously established method with modifications^{32 (link),33 (link)}. Briefly, mouse brains were quickly removed and washed with ice-cold PBS, and the SN was isolated from the right hemisphere. The SN was rapidly homogenized at 4 °C, and the mitochondria were pelleted through differential centrifugation. The final mitochondrial pellet was suspended in isolation buffer to yield a final protein concentration of approximately 1 mg/ml and immediately stored on ice. The protein concentration was determined by a Bradford assay. Then, 20 μg of mitochondria was used for the ATP assay.