Atp content assay kit

Glycogen contents in the LM were measured by a commercial kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) according to the anthracenone method. Amounts of ATP in the LM were determined through an ATP Content Assay Kit (Solarbio, Beijing, China) following the manufacturer's instructions. The concentrations of NAD⁺ and NADH in the LM were determined using commercial kits according to the manufacturer's instructions (SinoBestBio, Shanghai, China). The relative intracellular glycogen, ATP, NAD⁺, and NADH levels were calculated based on the quantified concentrations normalized to the weight of the sample.

Cellular ATP was measured by an ATP Content Assay Kit (Solarbio, Beijing, China) following manufacturer’s instructions. Cells were plated at ~10⁶ cells in 75 cm² culture bottles before the experiment. After treatment, cells were ultrasonically crushed and centrifuged at 10,000 g at 4 °C for 3 min. Collect the supernatant and detect ATP level. The content of ATP was determined by colorimetric method at 340 nm by Microplate Reader (Thermo Fisher Scientific, Inc. USA). Total ATP levels were reported as μmol/10⁶ cells.

Cells were seeded into six-well plates at a density of 1 × 10⁶ cells in 2 ml RPMI 1640 medium per well and cultured overnight. Cells were then treated with or without 10 μM KU60019 and maintained at 1% O₂ for 10–12 h. Glucose consumption, lactate production, ATP production, citrate production, succinate production, fumarate production, and PFKP and CS activity were detected using glucose assay kit (Solarbio® BC2500), LA assay kit (Solarbio® BC2230), ATP content assay kit (Solarbio® BC0300), citric acid (CA) content assay (Solarbio® BC2150), micro mitochondrial citric acid content assay kit (Solarbio® BC2175), succinate colorimetric assay kit (Sigma® MAK184), pyruvate (PA) assay kit (Solarbio® BC2200), acetyl-CoA assay kit (Solarbio® BC0980), fumarate assay kit (Sigma® MAK060), PFKP test kit (Nanjing Jiancheng Bioengineering Institute A129), and citroyl synthetase kit (Nanjing Jiancheng Bioengineering Institute A108) according to instruction of the manufacturers, respectively. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content.

ATP content was determined using ATP Content Assay Kit (Solarbio, Beijing, China) according to the instructions of the manufacturer. Mitochondria pellets were resuspended in 1 mL extraction buffer and centrifuged at 10,000× g for 10 min, after which 500 μL chloroform was added to the supernatant, and the mixture was centrifuged at 10,000× g for 3 min. The supernatant was collected for ATP detection.

The ATP content and synthase activity were measured in the anthers of the maintainer and C5-type CMS lines with an ATP content assay kit and a Na⁺K⁺-ATP synthase activity assay kit, respectively (Solarbio Co., Ltd., Beijing, China). The procedures were performed according to the manufacturer’s instructions (Solarbio, BC0300 and BC0065).

Cell density was determined by a microplate reader (Multiskan™ FC, Thermo Scientific). The ATP in cells was measured through an ATP Content Assay Kit (BC0300, Solarbio) as previously reported (Wang et al., 2019 (link)). D-Allulose, D-fructose, and glycerol were determined using a high-performance liquid chromatograph (HPLC, HITACHI) equipped with a refractive index detector (RID) monitor. Sugar-Pak™ I Column (85°C, Waters) was used with deionized water as the mobile phase at a flow rate of 0.5 ml/min.

Cells were seeded at approximately 3 × 10⁵ cells/mL in 6-well plates and treated with 2-DG (2 mM) alone for 24 h and buforin IIb alone (2 μM) or both for 12 h. After centrifugation, the culture medium and cells were collected to estimate the L-lactate and ATP content using the ATP content assay kit (Solarbio, Beijing, China) and L-lactate assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s protocol. Lactate dehydrogenase catalyzes the conversion of lactate to pyruvic acid, and the absorbance at 530 nm was determined spectrophotometrically after the addition of a color developing agent, which showed a linear relationship with lactate content. The collected cells were subjected to ultrasonic disruption and centrifuged at 8000× g for 10 min at 4 °C to collect the supernatant for intracellular ATP detection. Creatine kinase catalyzes the production of phosphocreatine from creatine and ATP. The level of ATP was determined by detecting the phosphocreatine content though the colorimetric method of phosphomolybdic acid at 700 nm. Six replicates were performed for each group, and the experiments were repeated three times to confirm the results.