Toxinsensor chromogenic limulus amebocyte lysate endotoxin assay kit

Human bronchial epithelial cell line BEAS-2B was obtained from the China Center for Type Culture Collection (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetra-zolium salt (MTS) was purchased from Promega (Madison, WI, USA). Fetal bovine serum was purchased from PAA (Linz, Austria). Ninety-six-well plates and cell culture dishes were purchased from Costar (Cambridge, MA, USA). A ToxinSensor chromogenic limulus amebocyte lysate endotoxin assay kit was purchased from GenScript (Piscataway Township, NJ, USA). Desferrioxamine (DFO) was purchased from Sigma-Aldrich.

DTT-SP₄, DTT-FD^mut, DTT-FD^wt, DTSP, DTSP^wt, and FD^mut expression vectors were transformed into Escherichia coli Rosetta (DE3) separately. After the bacterial culture reached an optical density (OD) of 0.6–0.8 at 600 nm in LB at 37°C, the protein expression was induced at 16°C for ~24 h using 0.2 mM isopropyl-β-d-1-thiogalactoside. Bacteria pellets were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS), and lysed by sonicating on ice. Cell debris was removed by centrifugation (12,000 × g for 1 h) at 4°C. The His-tagged recombinant protein was purified from the obtained supernatant using a 5-ml Ni-HiTrap affinity column (GE Healthcare) and eluted with PBS in the presence of 150–300 mM imidazole. The crude protein was further purified by gel filtration using a Superdex 75 column (GE Healthcare) with PBS. Freshly purified proteins were analyzed via 12% SDS-PAGE, then concentrated to ~2–5 mg/ml, and stored at −80°C for further use. The endotoxin levels in the purified proteins were reduced using Detoxi-Gel Endotoxin Removing Columns (Thermo Scientific, USA) before immunization. Endotoxin levels were quantified using a ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (Genscript, China). Endotoxin contamination levels of all proteins (1 μg/ml) used in this study were under the acceptance level (<0.1 EU/ml).

Serum LPS concentrations were measured with a ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript), following the manufacturer’s instructions. Briefly, samples were diluted 10- to 50-fold with endotoxin-free water, adjusted to the recommended pH, and heated for 10 min at 70 °C to minimize inhibition or enhancement by contaminating proteins. LAL reagents were added to serum and incubated at 37 °C for 45 min, and the absorbance was read at 545 nm. A spiked control at 0.45 EU/ml was performed for each sample to check that no significant inhibition or activation occurred.

Serum LPS concentrations were measured with a ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript), following the manufacturer’s instructions^{37 (link)}. Briefly, to minimize inhibition or enhancement by contaminating proteins, the samples (n = 9–10 per group) were diluted 10- to 50-fold with endotoxin-free water, adjusted to the recommended pH, and heated for 10 min at 70°C. To obtain an endotoxin stock solution, the lyophilized endotoxin standard was dissolved by adding 2 ml of LAL reagent water and mixed thoroughly for 15 min with a vortexer. LAL reagents were added to serum and incubated at 37°C for 45 min, and the absorbance was read at 545 nm. A spiked control at 0.45 EU per ml was included for each sample to check that no significant inhibition or activation occurred. The lower limit of detection (LLOD) was 0.01 EU per ml. The coefficient of variation equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent. The coefficient of variation absorbance was less than 10%.

Serum LPS concentrations were measured with a ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript), following the manufacturer’s instructions [83 (link)]. Briefly, to minimize inhibition or enhancement by contaminating proteins, the samples were diluted 10- to 50-fold with endotoxin-free water, adjusted to the recommended pH, and heated for 10 min at 70 °C. The lyophilized endotoxin standard was dissolved by adding 2 mL of LAL reagent water and mixed thoroughly for 15 min with a vortexer to obtain an endotoxin stock solution. LAL reagents were added to the serum and incubated at 37 °C for 45 min, and the absorbance was read at 545 nm. A spiked control at 0.45 EU/mL was included for each sample to check that no significant inhibition or activation occurred. The lower limit of detection (LLOD) was 0.01 EU mL⁻¹. The coefficient of variation (C.V.) equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent. The C.V. absorbance was less than 10%.