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).
Choline oxidase
Choline oxidase is an enzyme that catalyzes the oxidation of choline to betaine aldehyde, which is then further oxidized to betaine. This enzyme is commonly used in laboratory settings for the detection and quantification of choline in various biological samples.
Lab products found in correlation
2 protocols using choline oxidase
Characterizing ATX-Inhibitor Interactions
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).
Membrane Lipid Dynamics Analysis
Cluster analysis was performed using VutaraSRX cluster analysis tool over multiple images. GraphPad Prism was used to determine significance for raft size; CTxB (n=1,907), CTxB+mβCD (n=344), PIP2 control (n=3,076), PIP2+mβCD (n=778). Cell differentiation assay was performed single blind on images taken from multiple dishes (n=4 for each condition). All numbers are reported as mean±s.e.m. unless otherwise noted. As all samples were found to have a normal distribution, Student's t-test was used to determine significance with resultant P-values as reported.
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