Cls9018bc

To measure TAG content, we used a triglyceride extraction method as previously described (FOLCH et al. 1957 (link)). Six to ten replicates of six 3–5-day old flies were collected and washed in four dilutions (1:2,1:4,1:10,1:20) of isopropanol to remove excess food. The aqueous phase was used for glucose assays. The lipids were collected after extraction, evaporated under N₂ gas and reconstituted with 95% ethanol. Scintillant was added to samples with radioactive tracer instead of ethanol and analyzed on a beta counter (Beckman Coulter). For nonradioactive samples, samples were spun and placed into 96 well plates (Sigma-Aldrich, # CLS9018BC) and incubated with triglyceride reagent (200 µl; Thermo Fischer Cat #TR22421) at 37°C for 30 minutes. Precimat glycerol reagent (Thermo Fischer # NC0091901) was used as a standard. Total absorbance at 500 nm was measured in a plate reader (Beckman) and subtracted from a blank before determining the amount of triglyceride using the reference standard curve. All calculations were performed in Excel (Microsoft) and graphed in Prism.

To determine the amount of glycogen, we collected six replicates of six male flies and washed them in dilutions of isopropanol to remove food. Fasted flies were collected after 24 hours of starvation. Flies were homogenized in 1 M KOH for 30 seconds using steel balls and a tissue lyser (Resche MM400). Samples were heated for 30 minutes at 70°C. Saturated Na₂SO₄ was added prior to addition of 95% Ethanol for precipitation. The pellet was spun down and then reconstituted in water, heated at 70°C, before adding 95% Ethanol again. The pellet was centrifuged in the water/95% ethanol solution, the supernatant was removed and amyloglucosidase (Merck # A7420) was added overnight at 37°C. Samples were centrifuged and placed into 96-well plates (Sigma-Aldrich, # CLS9018BC) and incubated with glucose oxidase reagent (200 µl; Thermo Fischer, TR15221) at 37°C for 30 minutes. Glucose (1 mg/ml) was used as a standard. Total absorbance at 500 nm was measured in a plate reader (Beckman) and subtracted from a blank before determining the amount of glucose using the reference standard curve. All calculations were performed in Excel (Microsoft) and graphed in Prism.