Lal endotoxin quantitation kit

The PLO plasmid (pGS59) was a generous gift from Prof BH Jost (University of Arizona, USA), and the DS-PLO mutant plasmid (R219C/G85C) was a kind gift from Prof M Palmer (University of Waterloo, Canada). Proteins were generated as described previously^{16 (link),27 (link),28 (link)}. The abundance of proteins was measured by DC assay, and their purity evaluated using SDS-PAGE and Coomassie blue staining, as described previously^{16 (link)}.
A hemolysis assay was used to determine the activity of PLO, as described previously^{16 (link)}. Optical density (OD₄₅₀) was measured using a microplate reader (POLARstar Omega; BMG Labtech, Offenburg, Germany). Hemolytic units were mathematically determined by 4-parameter modelling using the Solver function in Microsoft Excel. Endotoxin concentrations in stock solutions of PLO were all <1 EU/ml, as determined by LAL assay (LAL endotoxin quantitation kit; Thermo Scientific, Hertfordshire, UK). To examine the potential binding to PLO, 1 μM atorvastatin, 10 μM alendronate, 10 μM zaragozic acid, 1 mM methyl-β-cyclodextrin, and 1 mM cholesterol (all Sigma) were incubated with 100 HU/ml PLO for 1 h, prior to conducting the hemolysis assay.

The plo plasmid (pGS59) was a gift from Dr H Jost (University of Arizona), and pyolysin protein was generated as described previously [14 (link), 62 (link)]. The activity of pyolysin was 628,338 HU/mg protein, as determined by hemolysis assay using horse red blood cells (Oxoid, Hampshire, UK), as described previously [14 (link), 63 (link)]. Endotoxin contamination was 1.5 EU/mg protein, as determined by a limulus amebocyte lysate assay (LAL endotoxin quantitation kit; Thermo Fisher Scientific, Hertfordshire, UK). Streptolysin O was purchased from Sigma, stored as 1 mg/ml solution, and activated with 10 mM dithiothreitol according to the manufacturer’s instructions (Sigma). To examine the potential for pyolysin binding to glutamine, 100 HU/ml pyolysin was incubated for 1 h in PBS with vehicle, 2 mM glutamine, or 1 mM cholesterol as a positive control, and a hemolysis assay was conducted.