First, we used the same FS-3 assay as described above, but instead of plasma 10 μL of human recombinant ATX (3, 10 and 30 nM) (Daresbury Proteins Ltd, Warrington, UK) was incubated with FS-3 substrate ± IND (30–300 μM).
In addition, the effect of IND on human recombinant ATX-induced hydrolysis of LPC 18:1 was assessed with the Amplex Red choline release assay. ATX was generated in-house, as described before [43 (link)]. Triplicate wells were loaded with 60 μL of reaction cocktail in ATX assay buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2, 30 μM BSA, pH 7.4), with of 10 μM Amplex Red, 1 U/mL horeseradish peroxidase (both from Thermo Fisher Scientific, Waltham, MA, USA) and 0.1 U/mL choline oxidase (MP Biomedicals, Irvine, CA, USA), resulting in an overall concentration of 100 μM LPC 18:1 (Avanti Polar Lipids, Alabaster, AL, USA) with 10 nM ATX ± IND (30–300 μM). The fluorescence (λexcitation = 560 nm and λemission = 590 nm) was recorded every 2 min by a FlexStation 3 Multi-Mode Microplate Reader for 240 min (Molecular Devices, San Jose, CA, USA).
In addition, the effect of IND on human recombinant ATX-induced hydrolysis of LPC 18:1 was assessed with the Amplex Red choline release assay. ATX was generated in-house, as described before [43 (link)]. Triplicate wells were loaded with 60 μL of reaction cocktail in ATX assay buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2, 30 μM BSA, pH 7.4), with of 10 μM Amplex Red, 1 U/mL horeseradish peroxidase (both from Thermo Fisher Scientific, Waltham, MA, USA) and 0.1 U/mL choline oxidase (MP Biomedicals, Irvine, CA, USA), resulting in an overall concentration of 100 μM LPC 18:1 (Avanti Polar Lipids, Alabaster, AL, USA) with 10 nM ATX ± IND (30–300 μM). The fluorescence (λexcitation = 560 nm and λemission = 590 nm) was recorded every 2 min by a FlexStation 3 Multi-Mode Microplate Reader for 240 min (Molecular Devices, San Jose, CA, USA).