Atp assay kit

Cell apoptosis was detected using an Annexin V-FITC apoptosis detection kit (KeyGen, Nanjing, Biotech, China) and an adenosine triphosphate (ATP) assay kit (Beyotime, Shanghai, China). Cells were cultured in 6-well plates with 2 mL medium. After 24 h transfection, cells were collected in a 1.5 mL centrifuge tube. Before Annexin V-FITC apoptosis analysis, cells were washed three times and double stained with FITC-Annexin V and PI. Then, cell samples were analyzed using a FACSCanto II Flow Cytometer (Becton Dickinson, Trenton, NJ, USA). Percentages of early apoptosis and late apoptosis cells were counted and used as the cell apoptosis rate. The ATP concentration was evaluated using an ATP assay kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. The relative ATP level of experimental groups was normalized with the NC group.

To detect tissue ATP, 100 mg of AICAR and/or paraquat treated insects were placed in 1000 μL of lysis buffer and homogenized. The lysate was centrifuged at 12,000 ×g for 5 min at 4°C. The supernatant was transferred to a new 1.5-mL tube. According to the manufacturer’s instructions, the ATP content was determined using an ATP assay kit (Beyotime, Jiangsu, China) based on a bioluminescence technique. The protein content was determined according to the method described above at the same time. The relative ATP level was expressed as an ATP value/protein value [66 (link)]. To examine the effect of TcAK1 and TcAK2 phosphorylation on the ATP content of Sf9 cells under normal conditions, we transfected the Sf9 cells with the phospho-mimetic TcAK mutant vectors (pIZT-TcAK1 S129D T247D or pIZT-TcAK2 S145D), then the ATP and ADP contents of the cells were determined using ATP assay kit (Beyotime, Jiangsu, China) as described above and ADP Assay Kit (Colorimetric/Fluorometric) (Abcam, Cambridge, MA, USA) according to the protocol, respectively. The cells transfected with the WT TcAK1 vector (pIZT-TcAK1 WT) WT or WT TcAK2 vector (pIZT-TcAK2 WT) were used as the controls. The ATP and ADP levels were normalized for the number of cells [67 (link)], and the ratio of ATP to ADP was calculated. Each experiment was replicated three times.

Cell apoptosis was detected using an Annexin V-FITC apoptosis detection kit (Nanjing KeyGen Biotech, China) and an adenosine triphosphate (ATP) assay kit (Beyotime, China). Cells were cultured in six-well plates with 2 ml of medium. After 24 h of transfection, the cells were collected in a 1.5-ml centrifugation tube. Before Annexin V-FITC apoptosis analysis, the cells were washed three times and double-stained with FITC-Annexin V and PI. The cell samples were analyzed using a FACSCanto II flow cytometer (Becton Dickinson, United States). Percentages of early and late apoptosis cells were counted and used to calculate the cell apoptosis rate. The ATP concentration was evaluated using an ATP assay kit (Beyotime, China), according to the manufacturer’s protocols. The relative ATP levels of the experimental groups were normalized to that of the NC group. Additionally, the protein expression levels of cell survival-related genes (BCL2, BAX, and Caspase-3) were also measured using western blotting.