Dye Dilution Technique

Radioisotope-labeling was performed essentially as described^{4 (link)}. Briefly, adult flies were habituated on the experimental food for four days prior to testing, with a transfer to fresh food on the second day. On the fourth day, flies were transferred to the experimental medium supplemented with 1–2 μCi/ml [α-³²P]-dCTP. After 24 h, flies were transferred to empty vials and allowed to groom for ~15 min. Flies were killed by freezing, counted, and then assayed in 5 ml of scintillation fluid (ScintiVerse^TM BD Cocktail, Fisher Scientific) in a Multi-Purpose Scintillation Counter (LS 6500, Beckman Coulter). Flies fed non-labeled food were used as controls and their scintillation counts were subtracted from experimental readings. These counts were equivalent to background. Aliquots (10–20 μl) of the non-solidified food with the radioactive tracer were used to calculate food volumes from scintillation counts. For diets that are difficult to accurately pipette, taking the mass of scintillation vials before and after dispensing the aliquot provides highly accurate and reproducible calibrations. For each condition tested, 4–5 vials containing 10–20 flies each were typically used. For ¹⁴C studies, experimental medium was supplemented with [¹⁴C]-leucine or [1,2-¹⁴C]-choline to 0.5 μCi/ml food (final). Flies from ¹⁴C studies were processed for scintillation counting by adding 250 ml of a 1:1 mixture of 30% (v/v) H2O2: 60% perchloric acid and heating for 2 h at 70 °C in a glass scintillation vial. After cooling to RT, scintillation fluid was added and radioisotope was measured as described above. To measure excreted radiolabel, flies were housed in 20-ml scintillation vials and food was provided in the cap. Discarding the cap then allowed measurement of excreted radiolabel in the empty vials.
Food intake measurements on dye-labeled food were performed similarly to the radioactive tracer assay described above. After habituation on the experimental food for four days, flies were transferred at 1 hour past lights-on to identical media containing 1% (w/v) FD&C Blue #1. After 15 min, 1 h, or 4 h of feeding, feeding was interrupted by freezing the vials at −80 °C. Frozen flies were transferred to 1.7 ml Eppendorf tubes and homogenized with a motorized pestle (VWR) in 50 μl of 1× PBS + 1% Triton X-100. The use of detergent (0.01% Triton X-100 is sufficient) is critical for accurate dye measurements from homogenized fly samples. After centrifugation to clear the debris, the absorbance of the supernatant was measured at 630 nm (A₆₃₀) on a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Flies fed non-labeled food were used as controls and their A₆₃₀ values (typically negligible) were subtracted from experimental readings. Serial dilutions of an initial 10 μl aliquot of the non-solidified dye-labeled food added to 0.99 ml of 1× PBS + 1% Triton X-100 were used to generate a standard curve. After determining the equivalent dye concentration of each fly homogenate using the linear fit of the standard curve (R² was typically >0.99), consumption was calculated by multiplying with the homogenate volume (50 μl) and dividing by the number of flies per sample. Absorbance of the dye with unlabeled fly homogenates did not overlap, demonstrating that eye pigments do not interfere with the assay (Supplementary Fig. 1a). The use of detergents (Triton X-100) and buffer (1× PBS) is important for fully additive absorbance values, likely by minimizing pH effects on dye extinction coefficient and dye loss through adsorption to tube surfaces. For each condition tested, 10 vials containing 5 flies each were typically used.