Limulus amebocyte lysate kit

Conventional UHMWPE particles were a generous gift from Dr. Timothy Wright, Hospital for Special Surgery. Particles were isolated from knee joint simulator test supernatants according to established protocols.^{14 (link)} Following isolation particles were washed twice in ethanol (96%, 70%) and, after evaporation of ethanol, suspended in PBS for storage in −80°C. The particles tested negative for endotoxin using a Limulus Amebocyte Lysate Kit (BioWhittaker, Walkersville, MD). The mean diameter of the particles was 0.48 ± 0.10 μm as measured by electron microscopy. Alzet model 2006 (Durect Corporation, Cupertino, CA) osmotic pumps were loaded either with carrier solution (1% BSA-PBS); carrier solution with 15 mg mL⁻¹ UHMWPE particles; or carrier solution with UHMWPE particles and 10 μg mL⁻¹ mouse recombinant IL-4 (R&D Systems, Minneapolis, MN). 6-cm-long vinyl tubing (Durect Corporation), prefilled with the appropriate solution, was used to connect the pumps to the coated or noncoated titanium rods.^{22 (link)}

Conventional UHMWPE particles were a gift from Dr Timothy Wright (Hospital for Special Surgery, New York) and obtained from knee joint simulator tests and isolated according to an established protocol [18 (link)]. Frozen aliquots of the particles containing serum were lyophilized for 4-7 days. The dried material was digested in 5 M sodium hydroxide at 60 °C for 1h, and ultrasonicated for 10 min. The digested particle suspension was centrifuged through a 5% sucrose gradient at 40 K rpm at 10 °C for 3 h. The collected particles at the surface of the sucrose solution were incubated at 80 °C for 1 h and centrifuged again through an isopropanol gradient (0.96 and 0.90 g/cm³) at 40K rpm at 10 °C for 1 h. The purified particles at the interface between the two layers of isopropanol were harvested and the isopropanol was evaporated from the particle mixture then lyophilized until dry. Particles were then re-suspended in 95% ethanol which was evaporated completely. The particles tested negative for endotoxin using a Limulus Amebocyte Lysate Kit (BioWhittaker, Walkersville, MD). The mean diameter of the particles was 1.0 ± 0.1 μm (mean ± SE, averaged from 125 scanned particles) measured by electron microscopy. The measurement was done by Dr. Lydia-Marie Joubert in the Cell Science Image Facility at Stanford University.