Lactate assay

For measuring lactate synthesis in cells we used Lactate assay kit (Trinity Biotech, Jamestown, NY) and modified manufacturer protocol. Briefly, we added 10 μl of conditioned media collected from the cells to 200 μl of lactate reagent in 96-well plate and incubated for 15 min at RT. Lactate standard curve (0, 20, 80, and 120 mg/dL) was used for quantification. Light absorbance was measured at 540 nm. The results of the lactate assay were normalized to total protein measured by Bradford assay.

For measuring lactate synthesis in zebrafish embryos, 4 dpf larvae were homogenized in 100 mM Tris, 4 mM EDTA buffer (20 μl/20 fish), heated at 95°C for 5 min and centrifuged at 12,000 x g at RT. Ten microliters of the supernatants were used for lactate assay as described above. The results of the lactate assay were normalized to embryo number.