Lal endotoxin assay kit

Highly-purified lipopolysaccharide (LPS) from Escherichia coli 026:B6 was purchased from Sigma-Aldrich (St. Louis, Missouri) to stimulate TLR4, while Zymosan (TLR2 ligand) and Resiquimod (TLR7/8 ligand) where purchased from InvivoGen (San Diego, California). Polymorphprep™ was purchased from Axis-Shield (Oslo, Norway) to separate polymorphonuclear neutrophils (PMNs) from peripheral blood mononuclear cells (PBMCs). All monoclonal antibodies used for flow cytometry were acquired from Becton-Dickinson (BD) Biosciences (San Jose, California). To avoid estrogens in culture media, we used Hank's balanced salt solution (HBSS) containing Ca²⁺ and Mg²⁺, in addition to phenol-red free Roswell Park Memorial Institute medium (RPMI) 1640 containing L-glutamine medium and, charcoal stripped and dextran treated, fetal bovine serum (FBS) from Invitrogen Life Technologies (Paisley, UK).
Any possibility of endotoxin presence from external sources in reagents and assay material was excluded using the chromogenic LAL Endotoxin Assay Kit from GenScript (Piscataway, New Jersey).
The doses used for stimulation were those recommended by the manufacturer and tested to obtain a sub-maximal response on the dose-response curve.

The coding sequence of S1 domain (1–685 amino acids) or RBD (319–541 amino acids) of the Spike protein of SARS-CoV-2 were codon optimized, synthesized, and subcloned into the pCAGGS vector to generate a fusion protein with the Fc portion of human IgG1. Human Expi293F cells (Thermo Fisher Scientific) were transfected with the S1-Fc and RBD-Fc expression vector, supernatants harvested, and S1-Fc and RBD-Fc protein purified by affinity chromatography using a Protein A HP column (Cytiva). Control-Fc protein (#AG714; Merck) was purchased. Trimeric Spike (1–1208 amino acids) with 6 proline substitutions retaining the prefusion conformation (Spike-6P) were prepared as described previously^{32 (link)}. Recombinant B38-CAP protein was expressed in E. coli. The cell lysate was prepared and centrifuged at 13,000 × g for 15 min. The resulting supernatant was subjected to ammonium sulfate precipitation, anion-exchange chromatography with a Q-Sepharose Fast Flow column (1.6 × 10 cm; GE Healthcare), and gel filtration chromatography with a Superdex 75 pg column (2.6 × 60 cm; GE Healthcare). To exclude potential contamination of endotoxin, the eluates were further passed through a Polymyxin B column^{26 (link)}. All the proteins were confirmed free of endotoxin contamination (<0.1 EU/µg protein) with LAL Endotoxin Assay Kit (Genscript).

The endotoxin levels of the liver tissues were measured using the toxin sensor^TM chromogenic Limulus amebocyte lysate (LAL) endotoxin assay kit (Genscript, Nanjing, China) according to the manufacturer's instructions. All glassware, solutions, and surgical instruments used in the experiment were autoclaved at 121°C for 15 min. Around 30 mg liver tissues were homogenized in phosphate-buffered saline (PBS), then were centrifuged at 1,672 × g at 4°C for 10 min. 100 ul collected supernatant samples were used to measure the endotoxin concentration (EU/ml).

The remaining LPS on the titanium discs (n = 6) after different surface treatments was measured using a toxin sensor chromogenic limulus amebocyte lysate (LAL) endotoxin assay kit (GenScript, Piscataway, NJ, USA). LAL reagents were added to vials containing the treated titanium discs and an endotoxin standard. After incubation at 37 °C for 45 min, a chromogenic substrate solution (100 µL) was added and incubated at 37 °C for 6 min. A stop solution (500 µL) and color stabilizer (500 µL) were added and measured at 545 nm using a spectrophotometer.

Silica nanoparticles were purchased from Micromod Partikeltechnologie (Rostock–Warnemünde, Germany). Before use, the particles were sonicated for 5 min and vortexed for 1 min. Preparations of silica nanoparticles were checked for contamination with lipopolysaccharide by using an LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA).

Recombinant αsyn monomers were generated as previously described, with minor modifications [26 (link), 54 (link), 55 (link)]. Briefly, mouse αsyn was expressed in Escherichia coli BL21 (DE3) (BioDynamics Laboratory) and purified by ion exchange using Q Sepharose Fast Flow (GE Healthcare). Endotoxin from Escherichia coli was removed using the ToxinEraser™ Endotoxin Removal Kit (GenScript), and the endotoxin levels were confirmed to be below the detection sensitivity with LAL Endotoxin Assay Kit (Genscript). After dialysis against 5 mM Tris–HCl and the addition of 10 × PBS to make 1 × PBS solution, αsyn monomers (5 mg/ml) were incubated at 37°C with constant agitation at 1,000 rpm for 7 days. The solution was sonicated for 5 min with a Bioruptor (Sonicbio) before injection.