2.6 Flow cytometry
PBS supplemented with 2 % heat-inactivated FBS was used as the staining buffer for flow cytometry. PBMCs from healthy controls and patients with psoriasis were resuspended in the staining buffer for the extracellular staining of CD4, CD19, CD14, CD15, CD11b, and HLA-DR, as well as the subsequent intracellular staining of EBI3 and p35. Directly labelled isotype-matched rat anti-mouse antibodies (BioLegend) served as controls. For intracellular staining, cells were first stimulated with 50 ng/mL phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA), 1.0 μg/mL ionomycin (Sigma-Aldrich) for 2 h, and then cultured for another 4 h in the presence of monensin (2 μL, BioLegend, San Diego, CA, USA). After extracellular staining, the cells were fixed and permeabilised using the Fixation/Permeabilization Solution Kit (BD Biosciences, St. Louis, MO, USA), according to the manufacturer’s protocol, and then EBI3 and p35 staining were performed.
The spleens of treated mice were collected, homogenised, and suspended as single cells in red blood cell lysis buffer. Skin tissues from the treated mice were collected, homogenised, and suspended as single cells in collagenase IV solution. Cells were extracellularly stained with CD11b, Gr-1, Ly6G, and Ly6C for 30 min at 4 °C in the dark. Directly labelled isotype-matched rat anti-mouse antibodies (BioLegend) were served as controls. After washing twice with PBS, the cells were resuspended in 200 mL of PBS and analysed via flow cytometry. For intracellular staining, the cells were stained extracellularly and then fixed and permeabilised using Perm/Fix solution (BD Biosciences, Franklin Lakes, NJ) for 30 min at 4 °C. Finally, the cells were intracellularly stained with anti-iNOS and anti-IL-10.
The following antibodies were used: fluorescein isothiocyanate (FITC) anti-human CD4, Brilliant Violet (BV)421 anti-human CD19, BV421 anti-human CD11b, phycoerythrin (PE) anti-human CD15 (BD Biosciences, San Diego, CA, USA), FITC anti-human HLA-DR (BD Biosciences), allophycocyanin (APC) anti-human EBI3, PE anti-human p35, FITC anti-mouse CD11b, APC anti-mouse Gr-1, PE anti-mouse Ly6G, APC anti-mouse Ly6C,PE anti-mouse iNOS, and PE anti-mouse IL-10 (all unspecified antibodies were from BioLegend).