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.
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.