1 µl eluted sample from the centrifuged flies was diluted in 9 µl PBS, vortexed, centrifuged briefly, and heated at 70°C for 5 minutes to inactivate endogenous Trehalase. 2 µl of the heated sample was added to 200 µl of Glucose Hexokinase Reagent (Thermo Scientific TR15421) with or without Porcine Kidney Trehalase (1∶1000; Sigma-Aldrich T8778-5UN), incubated at 37°C for 16 hours, and the absorbance at 340 nm (A340) was measured on a SpectraMax M5. The trehalose concentration in the sample was determined by subtracting the glucose concentration from the total sugar concentration.
Glucose hexokinase reagent
Manufactured by Thermo Fisher Scientific
The Glucose Hexokinase Reagent is a laboratory product designed to quantitatively determine the concentration of glucose in a sample. The reagent contains the enzyme hexokinase, which catalyzes the phosphorylation of glucose to glucose-6-phosphate, and glucose-6-phosphate dehydrogenase, which then converts the glucose-6-phosphate to 6-phosphogluconate with the concurrent reduction of NAD to NADH. The amount of NADH produced is proportional to the glucose concentration in the sample and can be measured spectrophotometrically.
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2 protocols using glucose hexokinase reagent
1
Trehalose Quantification Assay
2
Comprehensive Blood and Tissue Analysis
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Blood chemistry was determined by an OPTI® CCA‐TS2 blood gas analyser using an E‐Cl Type cassette (OPTIMedical). Blood, urine, gut sac and intestinal content clinical chemistry were performed using commercially available assays that were modified to work with small volumes.48 , 49 The concentration of hormones was determined using the following commercially available assays: PTH (1‐84; Quidel Corporation), intact FGF‐23 (Quidel Corporation). Urinary creatinine was determined using Infinity™ Creatinine Liquid Stable Reagent (Thermo Fisher Scientific). Glucose and Pi concentrations were determined using commercially available assays (Thermo Fisher Scientific Glucose Hexokinase Reagent and Pointe Scientific Phosphorus Reagent, respectively). Na+ was measured by flame photometry (BWB Technologies Ltd.).
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