Glucose oxidase peroxidase reagent

Alloxan monohydrate 98%, glucose oxidase-peroxidase reagent, sucrose, starch powder, and magnesium chloride-6-hydrate [MgCl₂, 6H₂O] were acquired from Sigma-Aldrich, Steinheim, Germany, as for the α-glucosidase enzyme, and acarbose were imported from Sigma-Aldrich, China, furthermore, the α-amylase enzyme and phlorizin dehydrate were both purchased from Sigma-Aldrich, USA, and Pentobarbital was imported from France. Dimethyl-sulfoxide (DMSO), Sodium chloride (NaCl) and potassium chloride (KCl) were purchased from Sigma Aldrich, Riedel-de Haen, Denmark. Ether and glibenclamide have been both acquired from Casablanca and Oujda respectively in Morocco, as for the Calcium chloride dihydrate [CaCl₂, 2H₂O] was imported from Spain, Scharlau Chemie, s.a. The sodium bicarbonate [NaHCO₃] was purchased from Puerto Rico, (Farco Chemical Supply), and finally Sodium phosphate monobasic dihydrate [NaH₂PO₄ · 2H₂O] from Panreac Spain.

For l-lactate assay, cells were seeded at a density of 0.1×10⁶ cells in 10 mL of growth medium supplemented with 10% FBS in 100 mm tissue culture plates. After 16 hours of incubation in humidified 5% CO₂ incubator at 37°C, cells were treated with the compounds. The plates were further incubated for 48 hours under normoxia or hypoxia. After treatment, the spent medium was collected for measurement of extracellular lactate and glucose. l-Lactate was estimated in culture supernatants using lactate assay kit from Eton Biosciences, Inc. (San Diego, CA, USA; cat no 1200012002) as per the manufacturer’s instructions. A standard l-lactate assay curve was plotted, and the concentration of l-lactate in samples was estimated from the standard curve using softmax pro software (Molecular Devices LLC, Sunnyvale, CA, USA). d-Glucose was estimated in culture supernatants using commercial glucose oxidase–peroxidase reagent (Sigma-Aldrich Co. cat no G3660). The plates were read at 540 nm using spectramax spectrophotometer. A standard d-glucose assay curve was plotted, and the concentration of d-glucose in samples was estimated from the standard curve using softmax pro software (Molecular Devices LLC). The level of metabolites was calculated as metabolite level per million cells.