Atp assay kit

E. coli cells were cultured in M9 medium until early mid-log phase (OD₆₀₀, 0.8 to 1.0) and collected for the quantitative determination of intracellular ATP/ADP ratios and acetyl-CoA levels using the ATP assay kit (Solarbio, Beijing, China), ADP assay kit (Solarbio, Beijing, China), and acetyl-CoA assay kit (Solarbio, Beijing, China). Extraction and quantification for intracellular ATP/ADP ratios were carried out according to the ATP and ADP assay kit instructions, and the analysis of ATP/ADP ratios was performed with an Agilent 1260 series HPLC system. The analytical column was a Thermo 250-mm by 4.0-mm ODS-2HYPERSIL C₁₈ column. The intracellular acetyl-CoA level was analyzed according to previous publications (52 (link), 53 (link)).

Transfected cells were seeded into 12-well plates and cultured for 48 h. The supernatant was collected, and the glucose and lactate levels were measured using the Glucose Assay Kit (Solarbio, Beijing, China) and Lactate Assay Kit (Solarbio, Beijing, China), respectively. The intracellular ATP level was measured using the ATP Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions.

Three plants per replicate were harvested and weighed immediately after removing the roots. Leaf relative water content (RWC) was determined following the method of Xu [102 (link)]. Fresh shoots were weighed quickly to obtain fresh weight (FW). Shoots were then soaked in distilled water for 4 h, and turgid weight (TW) was measured. To measure dry weight (DW), the shoots were dried at 80 °C for 24 h. RWC was calculated as follows: RWC (%) = [(FW-DW)/(TW-DW)] × 100. Superoxide radical (O₂^·−) was detected using the superoxide radical kit R30343 (Yuanye, Shanghai, China).
Leaf samples were used to measure ATP content. Homogenate was centrifuged for 10 min at 8000× g at 4 °C. Absorbance of the collected supernatant was measured with an ultraviolet spectrophotometer at 700 nm. ATP content was measured using an ATP assay kit (Solarbio, Beijing, China) and was expressed as μmol∙g⁻¹ DW.

The culture was collected and immediately centrifuged for 10,000 rmp for 1 min at 4 °C. The collected cells were washed with TE buffer to determine the intracellular ATP concentration. The ATP content was tested using an ATP assay kit (Solarbio, Beijing, China) following the manufacturer’s protocols. The collected cells were washed once with ice-cold PBS to determine the intracellular NADP⁺/NADPH concentration and then 0.5 mL of NADP extraction buffer for NADP⁺ determination or 0.5 mL of NADPH extraction buffer for NADPH determination. Heat extracts were incubated at 80 °C for 5 min, 10,000 rmp was centrifuged at 4 °C for 10 min, and 200 µL of the supernatant was measured using an NADP⁺/NADPH content assay kit (Solarbio, Beijing, China) according to the manufacturer’s protocols.

Lactate and ATP levels were measured using the lactate production assay kit (Solarbio) and the ATP assay kit (Solarbio), respectively, following the manufacturer’s protocol.

ATP levels in GC cells were measured using an ATP assay kit (Solarbio Life Science, Beijing, China) according to the manufacturer’s instructions. To measure the levels of glycolytic or mitochondrial ATP production, 2 × 10⁵ BGC-823 or 1 × 10⁵ MKN45 cells were seeded into plates and incubated in media containing 100 nM oligomycin A (Selleck) or 10 mM pyruvate in the absence of glucose and glutamine for 5 h. After being washed with PBS, the cells were lysed and examined for ATP levels using an ATP assay kit according to the manufacturer’s protocol.