In vitro MHC class II and CD40 assays.

A total of 10⁵ BMDCs were stimulated with 20 μg/ml zymosan (Saccharomyces cerevisiae cell wall), 10⁶ trophic forms, and/or 10⁶ mixed P. murina organisms at 37°C in 5.0% CO₂ for 24 h in BMDC culture medium. Adherent cells were collected from the plate using TrypLE expression dissociation reagent. Cells were washed with PBS containing 0.1% bovine serum albumin and 0.02% NaN3 and stained with appropriate concentrations of fluorochrome-conjugated antibodies specific for innate immune cells (anti-CD11c clone N418, anti-IA^d clone AMS-32.1, and anti-CD40 clone 3/23). Antibodies were purchased from eBioscience (San Diego, CA) or BD Biosciences (San Jose, CA). Expression of these molecules on the surface of the cells was determined by multiparameter flow cytometry using an LSRII flow cytometer. One thousand CD11c⁺ events were routinely acquired and analyzed using FlowJo software.