Multiparametric Immune Cell Analysis

The HLN and spleen cells were stimulated with Leukocyte Activation Cocktail (BD Pharmingen, San Jose, CA, USA) and LPS 10 μg/ml (Sigma-Aldrich, St.Louis, MO, USA) for 5 h, followed by blocking with purified rat anti-mouse CD16/CD32 (2.4G2, BD Pharmingen) for 10 min at 4 °C. Cell surface staining was performed with PerCP-Cy5.5 conjugated CD4 (RM4-5) or PerCP-Cy5.5 conjugated CD19 (1D3, BD Pharmingen) as described previously3 (link). Cells were fixed and permeabilized using a fixation/permeabilization kit (eBioscience, San Diego, CA, USA) or BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution kit (BD Pharmingen) according to the manufacturer’s instructions. Then, cells were stained at 4 °C with Alexa Fluor 488-conjugated IFN-γ (XMG1.2), PE-conjugated IL-17A (TC11-18H10), Alexa Fluor 647-conjugated Foxp3 (MF23), or PE-conjugated IL-10 (JES5-16E3, BD Pharmingen). Stained cells were washed twice and resuspended in 1% paraformaldehyde-PBS. Analysis of cell marker expression was performed using a FACSCanto II system (BD, Franklin Lakes, NJ, USA). Dead cells were gated out depending on forward scattering (FSC) and side scattering (SSC). Cells were analyzed with Diva software.