Quantifying Blue Dye Consumption in Flies

Flies were raised on control food or one of the experimental foods for 3 weeks without FD&C blue dye #1 before being transferred to the same diet with blue dye from the hours of 4:00 PM-6:00 PM, which was the 2-h period at which the most food was consumed. After 2 h, flies were anesthetized and collected in groups of 4 to measure the amount of blue dye consumed. Flies with blue on the outside of the body were not used. Flies were homogenized in 200 μl of PBS buffer and centrifuged. The absorbance of the supernatant was measured at 630 nm using a microplate spectrophotometer (Molecular Devices) similar to Wong et al. (2009) (link). The amount of blue dye in the samples was calculated from a standard curve made by serial dilution of blue dye in PBS buffer along with homogenate from age-matched flies exposed to non-dyed food in order to correct for absorbance of homogenate alone.