Saccharomyces cerevisiae zymosan a

β-glucan particles were prepared by treating Saccharomyces cerevisiae zymosan A (Sigma) with a hot-alkali treatment to remove TLR agonists as previously described [75 (link)]. Briefly, a suspension of zymosan particles were initially prepared and the particles were then boiled in 10 M NaOH for 1 hour followed by 5 washes in sterile PBS. The glucan particles were then dried in a Savant SpeedVac vacuum concentrator. Dried glucan particles were loaded with various concentrations of ovalbumin (OVA) or the recombinant S. aureus proteins (with or without TRITC or FITC-labeling) as previously described [29 (link), 51 (link)]. Glucan particles (GPs) were kept as frozen aliquots and were sonicated in an ultrasonic water bath for 15 minutes prior to their in vitro or in vivo use. Empty-GPs were prepared the same as the loaded-GPs minus the addition of any protein antigen.

1 x 10⁶ BMDCs were plated in 24 well-plates (Nunc, Roskilde, Denmark) in complete RPMI medium. 5, 20 or 50 axenically maintained larvae were washed thrice and added directly into DC cultures. In another series of experiments, larvae were separated from BMDCs using trans-well inserts (0.4 μm, BD Falcon). Alternatively, larva ES products were used instead of whole larvae. Lipopolysacharide (LPS; 0.1 μg/ml, E. coli 0127:B8) was used as a positive control for DC activation. After 24h of DC stimulation at 37°C under 5% CO2, supernatants were collected for cytokine detection and cells were stained for flow cytometric analysis. For re-stimulation experiments, larvae were removed 24 h post stimulation and BMDCs were further stimulated with LPS (0.1 μg/ml, E. coli 0127:B8) for an additional 24 h. In some experiments, BMDCs were treated with different doses (0.5 μg/ml up to 50 μg/ml) of M. corti ES products or McH prior to, at the same time with, or 18 h after LPS stimulation. In several experiments different stimuli were used instead of LPS for restimulation i.e.: Zymosan A (Saccharomyces cerevisiae; 50 μg/ml, Sigma), Curdlan (50 μg/ml, Wako), Lipotechoic acid (LTA, 10 μg/ml, InvivoGen) or phorbol 12-myristate 13-acetate (PMA; 0.5 μg/ml, Sigma). Control BMDC wells were treated in a similar way without exposure to parasite larva samples.