Quantifying Autotaxin Activity Assay

ATX activity was measured in conditioned media (CM) of WT and ATX-KO B16-F10 cells or BALF from mice injected with WT or ATX-KO B16-F10 cells, as described in [18 (link)]. Briefly, the fluorogenic LPC analog, FS-3 (Echelon Biosciences, Salt Lake City, UT, USA) was used as a substrate for ATX. For measurement of ATX activity in culture medium, CM was collected after 18 h of incubation in serum-free medium, clarified by centrifugation, filtered through a 0.22-μm filter, and concentrated using an Amicon Ultra 30,000 Centrifugal Filter Unit (Millipore; Billerica, MA, USA). For measurement of the activity in biological fluids, BALF was rapidly stored at −80 °C after collection and thawed on ice just before the assay. Five hundred microliters of BALF was then concentrated to 150 μL using an Amicon Ultra 10,000 Centrifugal Filter Unit (Millipore; Billerica, MA, USA). Activity was measured by incubating 30 μL of cell CM or murine BALF with 2 μM FS-3 substrate and 10 μM BSA (Sigma-Aldrich) in assay buffer consisting of 50 mM Tris, (pH = 8.0), 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, and 1 mM MgCl₂. A 10 nM final concentration of purified recombinant human ATX was used as a positive control. Fluorescence was read every 2 min for 4 h at 37 °C at wavelengths of 485 nm and 538 nm using a FlexStation3 plate reader (Molecular Devices, San Jose, CA, USA).