Fly Whole-Body Glucose Quantification

Whole-body total glucose was determined as described by [66 (link)]. Groups of five flies per sex were homogenized in 100 µL of 100 mM PIPES buffer (Sigma P6757, St. Louis, MO, USA) with porcine kidney trehalose at 5 µL per 2 mL (Sigma T8778) for 1 min at 6000 rpm by using a high throughput homogenizer (Qiagen, Hilden, Germany). Trehalose converts trehalose (present in the hemolymph) into glucose, so total available glucose levels were measured. The homogenates were then incubated at 37 °C for 1 h, and 10 µL was transferred to 100 µL of the Glucose GO assay kit (Sigma-Aldrich, St. Louis, MO, USA). The reaction mixture was incubated in an Environ Shaker at 37 °C for 10 min, and glucose was measured at OD 340 nm by using the SpectraMax M2e instrument (Molecular Devices Corp.) in relation to deionized water.