Normal human serum (NHS) from healthy donors with no prior history of Borrelia spp. infection was purchased from the Heidelberg University blood bank, factor B-depleted NHS (NHS-B) from Complement Technology, Inc (Tyler, Texas). For heat inactivation of complement (hiNHS, hiNHS-B), sera were incubated at 56°C for 30 min. Serum sensitivity was assessed using B. burgdorferi isolate B313 [37 (link),38 (link)], after confirming OspC expression, using a SYTO9 (BacLight bacterial viability kit; Molecular Probes) fluorescence-based survival assay [38 (link)]. In brief, cells grown to mid-logarithmic phase were harvested, washed and ~2.5x106 spirochetes each were resuspended in 50 μl BSK-H medium supplemented with 8 μg of solubilized recombinant H6-Salp15, or BSA for control, with or without 2 μl of mouse anti-Salp15 IS or nonimmune mouse serum. After 1 h at 25°C, 50 μl of 50% NHS or hiNHS was added (final concentration 25% v/v) and incubated for 20 h at 30°C. In some experiments, NHS-B or hiNHS-B were employed instead. Thereafter, cells were washed in 0.9% NaCl, transferred to microtiter plates and processed as recommended by the manufacturer. Fluorescence intensities were measured in triplicate on a microtiter plate reader (Victor2 plate reader, Perkin Elmer).
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