Albumin bovine fraction 5

The interaction of AIM proteins with immobilised IgM-Fc pentamers were examined at 25 °C using a Biacore T100 SPR instrument (GE Healthcare, Little Chalfont, UK). IgM-Fc pentamers were covalently immobilised on a CM5 sensor chip (GE Healthcare) at pH 4.5 to 1000 resonance units using the amine coupling procedure at a flow rate of 10 μL/min. A reference flow cell, on which BSA (albumin, bovine, fraction V; Sigma) was immobilised, was used to record the background response, which was subtracted from each sample. Measurements were performed according to single-cycle kinetic analysis. Recombinant AIMs were diluted in a running buffer (Dulbecco phosphate-buffered saline [D-PBS (−)] pH 7.4; Nacalai Tesque) at a concentration ranging from 50 nM to 5 μM, and injected into flow cells at a flow rate of 30 μL/min for 120 sec repeating five times at 60-sec intervals. After injection, AIMs were allowed to dissociate in the running buffer for 30 min. The results were analysed using Biacore T100 evaluation software. The differences in binding responses on the IgMFc-immobilised flow cell and the control BSA flow cell were fit to the heterogeneous ligand model to determine kinetic and affinity constants.

Cells were seeded on ultra-low attachment culture dishes (Corning, Corning, NY) in serum-free DMEM-F12 medium containing 50 μg/ml insulin (Sigma-Aldrich St. Louis, MO), 0.4% Albumin Bovine Fraction V (Sigma-Aldrich St. Louis, MO), N-2 Plus Media Supplement (Life Technologies, Grand Island, NY), B-27 Supplement (Life Technologies, Grand Island, NY), 20 μg/ml EGF (PeproTech Rocky Hill, NJ), and 10 μg/ml basic FGF (PeproTech, Rocky Hill, NJ) to support the growth of undifferentiated oncospheres. Cells were incubated in a CO₂ incubator for 1–2 weeks, and the numbers of oncosphere cells were counted under a microscope.

L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai which is a high-passage strain was provided by the National Institute for Control of Pharmaceutical and Biological Products in Beijing, China. The strain was cultivated at 28°C in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium supplemented with 5% albumin bovine fraction V (Sigma, USA) and 0.05% Tween-80 (Difco, USA).

Cells were cultured in ultralow attachment culture dishes (Corning, NY, USA) with serum-free DMEM-F12 medium containing insulin (50 μg/ml final concentration, Sigma-Aldrich, MO, USA), albumin (bovine) fraction V (0.4% final volume concentration, Sigma-Aldrich, MO), N-2 Plus Media Supplement (Life Technologies, NY, USA), B-27 Supplement (Life Technologies, NY, USA), EGF (20 μg/ml final concentration, PeproTech, NJ, USA), and basic FGF (10 μg/ml final concentration, PeproTech, NJ, USA) to sustain the growth of undifferentiated oncospheres. After the cells had been continuously incubated in a cell incubator for 1–2 weeks, cells in oncospheres were counted under a microscope.