Taurocholate tca

Villus cells were isolated from the ileum of the experimental animals by a calcium chelation technique as previously described [19 (link)] with few modifications. Briefly, isolated distal ileal villus cells were washed twice in TMA-HEPES buffer (50 mM KCl, 0.1 mM MgSO₄, 50 mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and 100 mM TMA-Cl) and suspended in 100 µL of the same buffer. Next, 10 μL of the cells from the suspension were added to 100 μL of reaction media containing 0.1 mM taurocholate (TCA; Sigma-Aldrich Corporation, St. Louis, MO, USA) with 100 μM ³H-taurocholate (PerkinElmer, Inc., Waltham, MA, USA), 50 mM KCl, 0.1 mM MgSO₄, 50mM Mannitol, 50 mM HEPES-Tris (pH 7.5), and either 100 mM TMA-Cl or 100 mM NaCl. The reaction was stopped at two minutes by adding 1 mL of ice-cold TMA-HEPES buffer. The stopped reaction mixture was filtered on 0.65 μm Millipore (HAWP) filter and washed twice with ice-cold stop solution. The reactions were carried out in triplicate for each of the two reaction mixtures. The filter was dissolved in 5 mL of scintillation fluid (Ecoscint A, National Diagnostics), and radioactivity was determined in a Beckman 6500 scintillation counter (Beckman Coulter Inc., Brea, CA, USA).

The plaque neutralization assay is based on the plaque assay described above with the following modifications. For neutralization, a fixed concentration of 1 × 10⁶ PFU of MNV was used. The serial dilution was conducted in 1× PBS in the presence of indicated additives. Specifically, bile acids (glycochenodeoxycholate GCDCA or taurocholate TCA, Sigma–Aldrich) were added to each dilution to a final concentration of 500 µM and purified antibodies (2D3, 4F9, or A6.2) were added to each dilution to a final concentration of 2 µg per ml. The mixtures (500 µl) were applied to the cell monolayer and the standard plaque assay protocol was followed.