Pe conjugated anti 1 a 1 e

Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated anti-F4/80, purified or PE-conjugated anti-CD21/35 (CR 2/1, clone 7E9) and PE-conjugated anti-I-A/I-E were purchased from BioLegend (San Diego, CA). FITC-conjugated Ly6G (RB6-8C5), PE-conjugated anti-Toll-like receptor 4 (TLR4) and PE-conjugated anti-CD64 were from BD Biosciences (San Jose, CA). U0126 was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany).

Iliac lymph nodes were excised from immunized mice and immediately immersed in Hank's Balanced Salt Solution medium and put on ice. Lymph nodes were then treated with 1 mg/mL Collagenase D (Roche Diagnostics, Indianapolis, IN) at 37 °C for 20 min to release cells. Cells were washed with PBS containing 2 mM EDTA, blocked with 1% normal rabbit serum, and stained with the following antibodies and reagents: Zombie Violet, biotinylated anti-Ly6G, biotinylated anti-Ly6C, Alexa Fluor 488 or PerCP-Cy5.5-conjugated anti-CD11c, PE-conjugated anti-I-A/I-E, PE/Cy7-conjugated anti-CD86, Alexa Fluor 488-conjugated F4/80 (all from BioLegend, San Diego, CA), and PE-conjugated anti-Siglec F (BD Bioscience, San Jose, CA). Biotinylated antibodies were detected with Alexa Fluor 488 or APC-conjugated streptavidin. After staining, cells were fixed in 2% paraformaldehyde. Cells were examined in a Canto II flow cytometer (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (FlowJo, Eugene, OR).