Cd11c pe cyanine5

For establishment of the airway allergy model, female C57 mice were chosen, and Alternaria alternata (Greerlabs, Lenoir, NC, 100 g/mouse in 50 μL) or PBS (50 μL) was administered intranasally on four consecutive days [27 (link)]. Four hours before the daily Alternaria alternata administration, 50 μg soluble rat-derived antigens of AC larvae (L4) or an equal volume of PBS treatment was administered via nasal drip. On the day after the last intranasal stimulation, mice were euthanized and BAL was harvested. Then, the cells derived from BAL were stained by Siglec F PE (BD), CD11c PE-Cyanine5 (eBioscience), or CD45 APC-cy7 (Biolegend) and analyzed by flow cytometry [28 (link)]. For the AC infection model, each Sprague-Dawley rat and BALB/c mouse were orally infected with 100 and 30 AC larvae (L3), respectively. AC larvae (L3) were collected from the tissue of infected Biomphalaria glabrata as previously described [29 (link)]. The mice and Sprague-Dawley rats were euthanized on days 7, 14, 21, and 28 after infection with AC.

Flow cytometry analysis was performed on a CytoFLEX S flow cytometer (Beckman Coulter).
For blood and brain macrophage and eosinophil assessment, PBMCs and BMNCs were isolated as previously described [25 (link)] and then incubated with Siglec F PE (BD), CD11c PE-Cyanine5 (eBioscience), or CD11b APC-cy7 (eBioscience) at 4 °C for 30 min.
To determine the cell sources of Chi3l3, BMNCs were stained with CD11b PE (eBioscience), F4/80 PE-Cyanine5 (eBioscience), CD45 PE-eFluor 610 (eBioscience), Ly-6C PerCP/Cy5.5 (Biolegend), CX3CR1 Alexa Fluor 488 (eBioscience), or CCR2 Phycoerythrin (R&D) at 4 °C for 30 min.
For measurement of IL-5 and IL-13 levels, spleen cells [30 (link)] from normal and AC-infected mice were incubated with CD3 APC (eBioscience), CD4 FITC (eBioscience), and IL-13 PE cy7 (eBioscience) at 4 °C for 30 min and analyzed by flow cytometry.
To determine the possible effects of Chi3l3 and sAg on IL-13 production, spleen cells isolated from normal and AC-infected mice were cultured in 24-well cell culture plates for 72 h, followed by a 12-h incubation with Brefeldin A and 6-h stimulation with PMA and ionomycin. Then, the cells were incubated with CD3 APC (eBioscience), CD4 FITC (eBioscience), or IL-13 PE cy7 (eBioscience) at 4 °C for 30 min and analyzed by flow cytometry.