Peritoneal Cell Thrombin Inactivation

Peritoneal cells from mMCP-4^+/+ mice and corresponding mMCP-4^+/− or ^−/− littermates (8–12 wk old) were collected by peritoneal washing with 10 ml of cold PBS, pH 7.4. Cells were centrifuged (300 g, 4°C, 10 min) and cultured in serum-free medium, HybridoMed DIF (Biochrom KG). The serum-free medium was supplemented with 50 μg/ml of gentamycin (GIBCO BRL). The cells were distributed in 24-well plates (Nunc; ∼0.5 × 10⁶ cells in 0.4 ml/well). Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. For activation of MCs, either the calcium ionophore A23187 (final concentration 2 μM), or anti–mouse IgE (2 μg) were added. To study the ability of the peritoneal cells to inactivate thrombin, 1 μg of thrombin (in 10 μl PBS) was added to the cell cultures. Samples from the conditioned media (50 μl) were taken at various time points and were frozen at –20°C. For analysis of residual thrombin activities, 20 μl of the samples were added to individual wells of 96-well microtiter plates, followed by the addition of 200 μl PBS and 20 μl of a 1.8 mM (in H₂O) solution of the chromogenic thrombin substrate S-2238. The absorbance at 405 nm was monitored with a Titertek Multiscan spectrophotometer (Flow Laboratories) and initial reaction velocities were determined with the DeltaSoft3 software.