Pe conjugated il 10 jes5 16e3

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; BD Pharmingen) or PerCP-Cy5.5 conjugated CD19 (1D3; BD Pharmingen) as described previously (9 (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 for 20 min at 4°C with Alexa Fluor 488-conjugated IFN-γ (XMG1.2; BD Pharmingen), PE-conjugated IL-17A (TC11-18H10; BD Pharmingen), Alexa Fluor 647-conjugated Foxp3 (MF23; BD Pharmingen), or PE-conjugated IL-10 (JES5-16E3; BD Pharmingen). Stained cells were washed twice in staining buffer (BD Pharmingen) 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 and side scattering. Cells were analyzed with FlowJo software.

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.