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).