Atp assay kit

ATP production was detected using the luminometric ATP Assay Kit (AAT Bioquest, Sunnyvale, CA) according to the manufacturer's instructions [41 (link)]. Briefly, 50 oocytes were seeded into each well of a 96-well white plate, and 200 μL of the ATP assay solution was added to the oocyte cultures. After gently mixing and incubating for 20 min at room temperature [42 (link)], the luminescence intensity was measured using the luminometer mode on a plate reader (Tecan, Zurich, Switzerland). The readings were normalized to the total protein content [43 (link)].

Ovarian cancer cell lines, IGROV-1 and SKOV3, were used in these experiments. IGROV-1 cells were grown in RPMI 1640 medium containing 10 % fetal bovine serum. SKOV3 cells were grown in DMEM/F12 medium with 10 % fetal bovine serum. These two cell lines were cultured with 1 % penicillin-streptomycin at 37 °C under a humidified atmosphere containing 5 % CO₂. NT1014 was provided by NovaTarg Therapeutics (Cary, NC). Metformin, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), and RNase A were purchased from Sigma (St. Louis, MO). All antibodies were obtained from Cell Signaling (Danvers, MA). The L-lactate assay kit was bought from Eton Bioscience (San Diego, CA). 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), caspase 3 assay kit, and ATP assay kit were bought from AAT Bioquest (Sunnyvale, CA). Annexin V FITC kit was purchased from BioVision (Mountain View, CA). Enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham (Arlington Heights, IL). All other chemicals were purchased from Sigma (St. Louis, MO).

ATP production was measured using the luminometric ATP Assay kit (AAT Bioquest, Sunnyvale, CA) according to the manufacturer's instructions [55 (link)]. Briefly, H9C2 cells were seeded in a 96-well white plate and 200 μl ATP assay solution was added. After mixing gently and incubating for 20 min at room temperature, luminescence intensity was measured using the luminometer mode on a plate reader (Tecan, Zurich, Switzerland) [56 (link)]. The readings were normalized to the total protein content.