Atp bioluminescent assay kit

ATP was measured in nontreated cells and upon treatment with inhibitors, as described in Drug Treatment section. ATP concentration was determined using adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit on GloMax 20/20 luminometer (Promega) and normalized to the protein content. In experiments with oligomycin, cells incubated with 0.1% DMSO (oligomycin solvent) were used as a negative control and ATP values from these cells were subtracted from ATP values obtained in oligomycin (+) experiments. For each set of measurements a second negative control (no cells) was included, and a background fluorescence was further subtracted from all other values.

Whole-body homogenized lysate (from 50 flies) was prepared in buffer D and cleared by centrifugation (500Xg, 30 s, 4 °C). ATP levels were determined using the ATP Bioluminescent Assay Kit (V6072, Promega) according to the manufacturer’s protocol. Photon emission was determined using a PerkinElmer counter. ATP contents were normalized to total protein amount.

Total cellular ATP levels were measured using a bioluminescent ATP assay kit (Promega, New Delhi, India) as per the manufacturer’s instructions. Luminescence was recorded with a microplate reader (Biotek Instruments, New York, NY, USA). For the quantitation of total nitric oxide synthesized by cells stimulated with different compounds for 24 h in the presence or absence of LPS, an assay measuring total nitrate released in the culture media was used. This colorimetric assay was performed using a commercially available kit as per the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA).

The microdialysis-intracerebral-guide cannula (Bioanalytical Systems, Inc., West Lafayette, USA) was implanted into the hippocampal DG zone (V 3.5 mm, AP 3.8 mm, ML 3.0). A microdialysis probe with a 2-mm membrane (Bioanalytical Systems, Inc., WestLafayette, USA) was then placed through the guide cannula into the DG region. Artificial cerebrospinal fluid (ACSF) with the ectonucleotidase inhibitor 6-N,N-diethyl-β-γ-dibromomethylene-D-adenosine-5-triphosphate FPL 67156 (ARL 67156 trisodium salt) was applied as the perfusate. The optimal perfusion flow-rate was set at 1 μl/min, and the sampling time for each rat was 2.5 h, 30 min after exposuring to the stressor of CUS. The ATP levels were determined using a bioluminescent ATP assay kit (Promega, Madison, WI, USA) and a luminometer (PerkinElmer) according to the manufacturer’s instructions. A calibration curve was obtained from standard ATP samples, and the luminescence of normal medium was considered to be the background ATP level.

Extracellular ATP was measured in the culture supernatants of different cells treated with doxorubicin for 48 h. A bioluminescent ATP assay kit (Promega, New Delhi, India) was employed as per the manufacturer’s instructions. Luminescence was recorded with a microplate reader (Synergy HT Biotek). To calculate the concentration of ATP released in the supernatant, an ATP standard curve of 0.78–500 nM range was employed.

ATP levels were determined using a bioluminescent ATP assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Total cellular ATP levels were measured using a bioluminescent ATP assay Kit (Promega, # G755A, G756A) as described in the manufacturer’s instructions. Luminiscence was recorded with an EnsightTM 96-well plate reader (Perkin Elmer). 3-(4,5-dimmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to estimate cell number following the manufacturer’s description (Tocris, # 5224), and absorbance was measured at 550 nm in an EnsightTM 96-well plate reader (Perkin Elmer).