Endotoxin free water

Highly pure rough LPS from E. coli K12 strain LCD25 was obtained from List biological labs. The FITC labelled LPS from E. coli 014 (Ra) and ³H/¹⁴C labelled LPS from Salmonella enterica sv. Typhimurium PR122 (Rc) were kind gift from Prof. Robert Munford (NIAID). Pooled normal human plasma was from Innovative Research. Endotoxin free water was from GE Healthcare Life Sciences. Fluorescein isothiocyanate (FITC), Alexa 488 hydrazide and Alexa 594 NHS were obtained from Molecular Probes. Purified apo-A1 from human plasma was obtained from Athens Research & Technology. The rabbit anti-apoA1 antibody was from Abcam. The TNF-α ELISA kit was from R&D. Chromogenic LAL Endotoxin Assay Kit was from Genscript. Recombinant human LBP protein was from R&D.

Because the PS nano-microspheres from Phosphorex Inc. were provided in an aqueous vehicle containing 0.1% of the detergent Tween, which alone could produce significant toxicity, dialysis was performed to remove it. Two milliliter samples of as purchased suspensions were loaded into 0.2 – 2.5 mL capacity 8 Kd MW cutoff Tube-O-DIALYZER™ micro dialysis tubes (Millipore Sigma, Burlington, MA) and floated in a large beaker containing 4 L of sterile deionized endotoxin-free water (GE Healthcare, Boston, MA) for 48 h at 4 °C with gentle stirring. Three 2.0 mL samples were dialyzed together, which would be expected to result in a dilution of the vehicle contaminant by a factor of 6/4000 = 0.0015, resulting in a negligible final Tween concentration of 0.00015%. At the end of dialysis the dialyzed test MNP suspensions were removed and their volumes, which increased slightly due to the greater osmotic pressure of the MNP suspension relative to the water, were measured in order to adjust the concentration of the samples. The dialyzed samples of PS MNPs from Phosphorex as well as the original non dialyzed carboxyl-PS MNPs from Thermo were then diluted to the desired starting MNP concentration and vortexed for 20 seconds.