Autokit glucose reagent

Following a 6-hour fast, time 0 blood was collected via tail bleed followed by an intraperitoneal (i.p.) injection of glucose (2 g/kg for control diet and 1.3 g/kg for HFD). Different amounts of glucose were used for the control-fed and HFD groups because HFD-fed animals have a lower percent lean body mass as compared to total body weight thus potentially biasing results towards showing impaired glucose tolerance in the high-fat group [54 (link)–56 (link)]. Regardless, statistical comparisons were only made between mice that received the same amount of glucose per body weight. Tail blood was then collected into 300K2E microvette EDTA tubes (Sarstedt) over the course of 120 min and then centrifuged at 3000 rpm for 30 min at 4°C for the separation of plasma. Plasma glucose was then measured using the Autokit Glucose Reagent (WAKO) per manufacturer's instructions. For insulin tolerance tests (ITTs), mice were fasted 6 h. Time 0 blood was obtained via tail bleed followed by an intraperitoneal (i.p.) injection of insulin (at 0.75 units/kg). Tail blood was collected and plasma glucose analyzed as described above.

Feeding larvae washed out from culture vials were collected in 630 µm mesh-fitted baskets (Genesee) and rinsed to get rid of adherent food particles. Larvae were dried and divided onto into piles (10–12 larvae each) on a strip of parafilm. Larvae were bled by tearing the cuticle with Dumont 5 forceps (Electron Microscopy Sciences). Two µl of colorless hemolymph was aspirated from each pile and separately transferred to 96-well plates (Thermo-Scientific) containing 0.1% N-Phenylthiourea (Sigma-Aldrich) in 50 µl PBS. 150 µl of Autokit Glucose reagent (Wako) was added to each well and incubated at room temperature for 20 min before measuring absorbance at 505 nm. Glucose concentration was calculated from a standard curve generated with manufacturer’s glucose standards. For trehalose assays, 8 µl of dilute hemolymph was treated with 5 µl of (diluted 8 X) porcine kidney trehalase (Sigma) overnight at 37 °C. Ten µl of treated sample was assayed for trehalose as described for glucose.

Larvae were divided into 4 piles (10–12 larvae each) on a strip of parafilm. Larvae were bled by tearing the cuticle with Dumont 5 forceps (Electron Microscopy Sciences). Two microlitres of colorless hemolymph was aspirated from each pile and separately transferred to 96-well plates (Thermo-Scientific) containing 0.1% N-Phenylthiourea (Sigma-Aldrich) in 50 µl PBS. 150 µl of Autokit Glucose reagent (Wako) was added to each well, and incubated at room temperature for 20 min before measuring absorbance at 505 nm. Glucose concentration was calculated from a standard curve generated with manufacturer’s glucose standards. For trehalose assays, 8 µl of dilute hemolymph was treated with 5 µl of (diluted 8X) porcine kidney trehalase (Sigma) overnight at 37 °C. Ten microlitres of treated sample was assayed for trehalose as described for glucose.