Atp detection assay kit

Pyruvate levels were measured using the Pyruvate Assay Kit (Cayman) following manufacturer’s recommendation. Briefly, the cells were washed with PBS and then centrifuged at 10,000×g for 5 min. The supernatant was discarded, 0.5 ml of 0.25 M metaphosphoric acid (MPA) added to the cell pellet and placed on the ice for 5 min to deproteinate the sample. The suspension was centrifuged at 10,000×g for 5 min, the supernatant removed and 25 μL of potassium carbonate added to neutralize the acid. After centrifugation at 10,000×g for 5 min to remove any precipitated salts, supernatant was discarded. The sample was diluted in 1:2 with assay buffer that was subjected to microplate reader analysis at 590 nm. ATP levels were determined using ATP Detection Assay Kit (Beyotime Biotechnology) according to manufacturer’s instructions. Enough standard ATP solution was prepared and added to each well to exhaust the background. Subsequently, 20 μL of sample or standard was added to each well and a luminometer was used to measure the relative light unit (RLU) value. The ATP levels of samples were calculated by referring to the standard curve under the same conditions

Half cortex of male offspring was isolated and homogenized with Lysis buffer (0.32 M Sucrose, 5 mM CaCl₂, 3 mM Mg(Ace)₂, 0.1 mM EDTA, 10 mM Tris–HCl, 0.1% NP40), followed by centrifugation at 12000 g at 4 ℃ for 5 min. The supernatant was used for ATP measurement using ATP Detection Assay Kit (Beyotime, S0026). TECAN Infinite M200 illuminometer was used to record the luminescence.

The Lactate dehydrogenase (LDH) assay kit (C0016, Beyotime Biotechnology) and L-Lactate Assay Kit II (1200051002, Eton Bioscience) were used to measure intracellular LDH and lactate levels, respectively, in DLBCL cells according to the manufacturer’s protocols. ATP generation was detected using an ATP detection assay kit (S0026, Beyotime Biotechnology).