0 μg/ml rPnxIIIA with or without 1.0 μg/ml anti-CD11a rat MAb (Abcam, Cambridge, UK), which cross-reacts with the α subunit of mouse CD11 as a neutralizing antibody, was measured after 2, 6, 12, and 24 h of incubation as described above. To assess the cytotoxicity of P. pneumotropica reference strains toward J774A.1 cells, a whole bacterial cell cytotoxicity assay was performed briefly according to the methods of Kehl-Fie et Ulixertinib datasheet al. . The results were reported as the percentage of LDH released from all the lysed cells. The experiments were repeated in triplicate.
ECM-binding assay ELISA was used to determine the binding of rPnxIIIA variants to components of rodent ECMs. In brief, a 96-well microtiter plate coated with rat collagen type I (BD BioCoat, BD) was used for the binding assay of rat collagen type I, and rat collagen type II (Chondrex, Redmond, WA, USA), mouse collagen type IV (BD), and mouse laminin (BD) were differently selleck chemical coated on a 96-well microplate (As one, Osaka, Japan) according to the manufacturer’s instructions. ELISA was performed with a protein detector AP microwell kit (KPL, Gaithersburg, MD, USA), anti-6× Histidine
tag monoclonal antibody (Biodynamics laboratory), and rPnxIIIA variants, the concentrations of which were adjusted to 0.5-50 μg/ml of the final concentration. For the whole-cell binding assay of P. pneumotropica reference strains, precultured cells were adjusted the OD reached 1.0 and incubated on a 96-well microtiter plate coated with rat collagen type I (BD) at 37°C for 24 h. Thereafter, the plate was briefly washed and stained
with 0.1% safranin, according to the method of Davey and Duncan . The quantification of adherent cells was determined by measuring the A490 with a plate reader. Hemagglutination and hemolytic assay Defibrinated sheep blood was obtained from Nippon Bio-Test Laboratories (Tokyo, Japan) and washed 3 times with sterilized phosphate-buffered saline (PBS) prior to conducting the assays. Hemagglutination activity was determined in V-cut bottom 96-well microtiter plates (Corning, Horseheads, NY, USA). Fifty micro milliliters of diluted rPnxIIIA variants or overnight cultures of P. pneumotropica reference strains were added to wells containing 2% sheep erythrocytes. The plate was incubated at RT for Morin Hydrate 1 h, and thereafter, the plate was imaged and visualized with a GeneGenius Bio Imaging Selleckchem PSI-7977 System (Syngene, MD, USA). A hemolysis assay was performed according to a previously described method  that used 2% sheep erythrocytes. Generation and purification of rabbit antisera In brief, crude rabbit antisera against the entire rPnxIIIA and rPnxIIIE proteins were prepared using the methods of Schaller et al. . The crude antisera were further purified on an HiTrap rProtein A FF column (GE Healthcare) mounted on an FPLC device, and rabbit IgG was prepared for immunological experiments.