Plates were then washed four times with PBS containing 0.05% Tween-20. Serum sample were diluted 1:300 in PBS and a threefold dilution series www.selleckchem.com/products/PLX-4032.html was performed. A total of 100 μL per well of the serum dilution was transferred to the LCMV-coated plates. After 1 hour of incubation at room temperature, plates were washed four times, followed by incubation with 100 μL per well of HRP-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch) diluted 1:30 000 in PBS, followed by 1 hour incubation. Thereafter, plates were again washed four times and 50 μL per well of the peroxidase substrate OPD (SIGMA) were applied
and the color reaction stopped after 10 min by adding 100 μL per well of 2 M sulfuric acid. OD was determined at a wavelength of 492 nm. LCMV-specific Ab titers were determined by an endpoint titer 0.1 OD over background. To determine the viral antigen specificity of these Abs, cell lysates of LCMV-infected and noninfected B16 melanoma cells find more were immunoprecipitated with IgG from LCMV immune serum that were bound to protein G-coupled sepharose (GE Healthcare). Samples were separated by 4–12% gradient SDS-PAGE (SERVA) and visualized with rabbit anti-LCMV serum
(1:5000), followed by HRP-conjugated donkey anti-rabbit IgG (Dianova). The ECL plus detection system (GE Healthcare) was applied for visualization. Single-cell suspensions of splenocytes were obtained by mechanical disruption. IFN-γ production of CD8+ T cells was determined by intracellular IFN-γ staining (anti-IFN-γ; clone XMG 1.2, ebioscience) after restimulation of 106 splenocytes with 10−7M LCMV GP33 peptide or LCMV NP396 peptide in the presence of 10 μg/mL Brefeldin A (SIGMA). CTL- and NK-cell activity was determined in a 51Cr-release assay. Target cells were loaded with 51Cr for 2 hours at 37°C and then incubated for 5 hours at 37°C with splenocytes that were previously titrated in a threefold
dilution series. Duplicate wells were assayed Amoxicillin for each effector-to-target ratio and percentages of specific lysis were calculated. Data were analyzed using SigmaPlot Version 9.0 software. Significant differences were evaluated with Mann–Whitney U-test using InStat3 software (GraphPad). The authors thank Maike Hofmann for helpful discussions and critical comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft DFG (Pi295/6-1 to H.P. and SFB490 to A.W.). The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.