Lithium heparin

All animal experiments were performed in compliance with the guidelines established by the USC Institutional Animal Care and Use Committee. Rats were administered doses of 10 nanomoles of rhodamine covalently linked to the amino terminus of recombinant L4F-A192. Rats were administered fluorescein L4F, at doses up to 50 nanomoles. L4F was chemically synthesized as follows, Met-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Phe-Lys-Glu-Ala-Phe-Leu-FITC (LifeTein, South Plainfield, NJ). Rats were bled at indicated time points from the tail into plasma separator tubes with lithium heparin (Becton, Dickinson and Co, Franklin Lakes, NJ). Serum was collected by spinning down blood samples and collecting supernatant. Plasma concentrations were determined using a calibrated fluorescence microplate assay run on a BioTek Synergy H1 Hybrid Multi Mode Microplate Reader (Biotek, Winooski, VT).

On day 0, P. leucopus animals were each injected subcutaneously with B. burgdorferi strain B31 recombinant lipidated OspA (lot # 42537 from Aventis Pasteur, Swiftwater, PA) (Pal et al., 2003 (link); Tsao et al., 2004 (link)) at a dose of 0.45 μg and keyhole limpet hemocyanin (Sigma–Aldrich, St. Louis, MO) (Martin et al., 2007a ) at 1.4 μg per gram of animal mass and in a volume of 100 μl of the following buffer: 50 mM Tris, pH 7.5–10 mM NaCl-0.3% Triton X-100. The purity and the absence of proteolysis of the recombinant OspA was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoreis. Protein concentrations were determined with a Microplate BCA Protein Assay Kit (Pierce, Rockford, IL). On day 21, P. leucopus were each intraperitoneally injected with 10⁴ cells of B. hermsii in infected plasma. Animals were euthanized on day 28 by CO₂ overdose, followed immediately by cardiocentesis and cervical dislocation. Blood was collected into 1.7 ml tubes coated with lithium heparin (Becton-Dickinson, Franklin Lakes, NJ). Plasma was recovered by serial 5 s centrifugations at 3000 × g in a microcentrifuge and then pooled. The liver, spleen, and heart were removed intact by dissection and weighed. Whole blood specimens and spleens were kept frozen at −80° C until DNA extraction. The dissected livers were fixed in 10% neutral-buffered formalin solution.