Atp colorimetric assay kit

NAM, sorbitol and sorbitol dehydrogenase were purchase from Sigma (St Louis, MO). The NAD/NADH cell based assay kit was purchased from Cayman (Ann Arbor, MI). The ELISA kit for NAMPT was purchased from Adipogen (San Diego, CA). The ATP colorimetric assay kit and lactate assay kit were purchased from Abcam (Boston, MA).

RVLM tissues were centrifuged at 10,000× g for 10 min after homogenization in a protein extraction solution (Pierce, Rockford, IL, USA), ATP concentration in the supernatant was determined by an ATP colorimetric assay kit (AbCam, Waltham, MA, USA) using a microplate reader (ThermoFisher Scientific). The ATP level was normalized to the protein concentration of the sample. Each measurement was performed in triplicate and the mean was used for statistical analyses.

Tissues were homogenized on dry ice using the ice-cold ATP assay buffer provided as part of the ATP colorimetric Assay kit (Abcam, Cambridge, UK, cat# ab83355;). A 5 μL aliquot of the homogenized tissue was used to determine protein concentrations using the Thermo Scientific BCA method. ATP concentrations in the samples were calculated by plotting the measured optical densitometry at 570 nm in a microplate reader versus the linear distribution generated by the standard curve, with a final adjustment for protein concentration.

An BioVision ATP colorimetric assay kit was used to quantify ATP levels in human astrocytes. Cells were cultured on a 60-mm dish (Corning) and were transfected with the indicated si-RNAs followed by adding water or KRGE (0.5 mg/mL) for 24 h in DMEM without FBS. After trypsinization, the cells were moved to Eppendorf tubes. The cell pellet was lysed in ATP assay buffer (140 μL) and incubated for 5 min at room temperature. The Eppendorf tubes were centrifuged at 15,000 rpm for 5–10 min at 4 °C. Next, the supernatant was mixed with the reaction mixture reagents (1:1 ratio, total 100 μL) on 96-well plates (Corning). After incubation (0.5–2 h) without light at room temperature, absorbance (570 nm) was measured using Epoch Microplate Spectrophotometer (BioTek). Lysed cells were quantified using BCA reagent plus copper sulfate. ATP levels (nmol ATP/mg protein) in the water-treated control group were determined as “1”, and the KRGE-treated groups were calculated to the control group.