Lal assay

Recombinant HARS (amino acids 1–506) was expressed as a soluble protein in E. coli without tags. HARS (1–506) was purified from lysed E. coli cells by anion exchange chromatography (Q-Sepharose HP) at pH 7.4 with elution by a gradient of increasing NaCl concentration, followed by hydrophobic interaction chromatography with phenyl Sepharose HP at pH 7.0 using a reverse gradient of ammonium sulfate. The protein was further purified using ceramic hydroxyapatite chromatography at pH 7.0 and was eluted by an increasing sodium phosphate concentration, after which the protein was buffer exchanged into 20 mM sodium phosphate, 150 mM NaCl pH 7.0, and sterile filtered for storage at −80 °C. Purity was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and size-exclusion chromatography, which demonstrated > 95% purity and less than 3% high-molecular weight material. The endotoxin level was determined using an LAL assay (Charles River) and shown to be less than 1 EU/mg.

Endotoxin content in SWCNT and GO dispersions was assessed using the chromogenic limulus amoebocyte lysate (LAL) assay (Charles River Endosafe, Charleston, SC). The endotoxin content was found to be below FDA-mandated limits of acceptance (0.5 EU/mL) (data not shown). To verify these results, SWCNT samples were also assessed using the TNF-α expression test (TET) that enables unequivocal detection of endotoxin with a sensitivity that is comparable to the conventional LAL assay, but without any interference with the assay, as described previously^{18 (link)}. In brief, HMDM were exposed to nanomaterials or lipopolysaccharide (LPS) in the presence or absence of the specific LPS inhibitor, polymyxin B (10 µM) and TNF-α secretion was measured at 24 h of exposure using a Human TNF-α ELISA Kit purchased from Abcam (Sweden).