Atp assay kit

ATP (mol/g protein) was measured in the cardiac tissue samples using ATP assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Fresh LV tissues were harvested, washed in cold PBS, frozen promptly and used on the same day for determination of content of ATP and lactate. ATP assay was performed using commercial ATP assay kits (Lot number A095-2-1, Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instruction. ATP assay was based on the fluorescence signal emitted during luciferin catabolism by luciferase, with fluorescence intensity being proportional to the content of ATP. Fluorescence of standards and test samples was read using a Victor^TM X2 multimode plate reader (PerkinElmer) luminometer (Waltham, USA). Lactate content in heart tissues was measured by colorimetry using a commercial L-lactatic acid colorimetric assay kit (Lot number: E-BC-Ko44-M, ELAB science - BioScientific Pty. Ltd. (Sydney, Australia)). Using NAD⁺ as H⁺ receptor, LDH catalyzes the reaction of lactic acid and NAD⁺ to generate pyruvic acid and NADH, respectively. NADH was subsequently converted to NBT, which is a purple chromogenic substrate. OD values at 530 nm were measured using the Victor^TM X2 multimode plate reader, and the concentration of lactic acid calculated based on the standard curve. Samples were determined in duplicates and the average was reported.

To detect the impact of FANS-RNAi on glucose metabolism, the levels of ATP and lactic acid were detected using the appropriate kits. ATP assay kits and lactic acid assay kits were purchased from Nanjing Jian-cheng Bioengineering Institute (Nanjing, China). Briefly, after A549 and NCI-H1299 transfection by FASN-siRNA, six groups of cells were harvested and homogenized in the cell lysates. The standard sample was diluted by a gradient to make a standard curve using a fluorescence microplate reader (BioTek model FLx800, CA). The ATP and lactic acid computation formulas are as follows: $$\mathit{ATP}=\frac{\text{ODtest}-\text{ODcontrol}}{\text{ODstandard}-\text{ODero}}\times \text{standard sample}\times \frac{\text{sample dilution multiple}}{\text{protein concentration}}\left(\text{grot}/\mathrm{L}\right)$$ $$\text{Lactic acid}=\frac{\text{ODtest}-\text{ODzero}}{\text{ODstandard}-\text{ODzero}}\times \text{standard sample}\times \text{sample dilution multiplier}.$$
The experiments were performed in triplicate.

The Ang II concentration in the serum and heart was measured using an Ang II enzyme-linked immunoassay (ELISA) commercial kit (Bioswamp, Wuhan, China). ATP levels in myocardial tissues were measured using ATP assay kits (Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.

To determine the functions of the mitochondria in neurons, mitochondria-related indices such as ROS production, mitochondrial membrane potential (MMP, ΔΨm), ATP levels, and GSH levels were evaluated. ROS levels were evaluated using the Reactive Oxygen Species Assay kit (Beyond, Shanghai, China), mitochondrial membrane potential was determined using the JC-1 staining kit (Beyond, Shanghai, China), ATP levels were evaluated using the ATP assay kit (Jiancheng, Nanjing, China), while GSH levels were measured using the GSH assay kit (Jiancheng, Nanjing, China), following the manufacturer’s instructions.