Glycin

Culture supernatants from four to five replicate pellets per group and time point were pooled, and 100 µL was incubated with 100 µL of substrate solution [10 mg/mL p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M Glycin (Carl Roth, Karlsruhe, Germany), 1 mM MgCl₂, and 1 mM ZnCl₂ (both from Sigma-Aldrich, St. Louis, MO, USA), pH 9.6]. After 120 min, the absorbance was recorded at 405/490 nm (FLUOStar OMEGA, BMG LABTECH, Ortenberg, Germany). The substrate conversion was referred to a p-nitrophenol-derived standard curve (Sigma-Aldrich, St. Louis, MO, USA) and calculated as ALP enzyme activity (ng/mL/min).

DON was obtained from Romer Labs (Tulln, Austria) and CER was purified as previously described [28 (link)]. Cell culture media and supplements were obtained from Gibco® Life Technologies (Karlsruhe, Germany). KH₂PO₄, KCl, NaCl, Na₂HPO₄, D-glucose, HEPES, CaCl₂, MgCl₂, glycin, formaldehyde, sodium dodecyl sulfate (SDS), NaOH and Triton X-100 were purchased from Carl Roth (Karlsruhe, Germany) and Lucifer Yellow CH di-lithium salt from Santa Cruz Technologies (Dallas, TX, USA). For LC-MS/MS measurements, MS grade water was purchased from VWR (Fontenay-sous-Bois, France), acetonitrile (ACN) and methanol (MeOH) from Honeywell (Seelze, Germany). Acetic acid was obtained from Merck (Darmstadt, Germany) and ammonium acetate (both LC-MS grade) from Sigma Aldrich (Vienna, Austria). DON-3-sulfate, DON-15-sulfate and DON-3-glucuronide were kindly provided by Dr. Philipp Fruhmann (Technical University of Vienna) and synthesized as previously described [44 (link)].