Fluorometric Zebrafish Glucose Assay

Glucose measurements were performed 3 times on 10 zebrafish larvae per condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) [26 (link)]. The larvae were collected in 1.5 ml microcentrifuge tubes. Excess medium was removed and embryos were frozen on crushed dry ice. After thawing, 200 μl PBS was added and the larvae were homogenized using a hand-held mechanical homogenizer. Reactions were assembled on ice in black, flat bottom 96-well plates (Costar). Standard curves were generated using glucose standard solution (according to instructions) and were included in each assay. To measure glucose in embryo extracts, 15 μl of sample were used. Control reactions without sample lysate were included in each row. Reactions were incubated for 30 minutes at 37°C in the dark. Fluorescence (excitation 535 nm; emission, 590 nm) was measured using a Safire II plate reader equipped with XFLUOR4 software (v 4.51). Fluorescence values were corrected by subtracting measurements from control reactions without sample. Glucose levels were interpolated from standard curves. Each sample was measured in triplicate and each experiment repeated three times.