Anti cd14

To determine the frequency of cells expressing CD14 and CD16, peripheral blood mononuclear cells (PBMC) (500,000 cells) were obtained from heparinized blood and stained with anti-MHC II, anti-CD14 and anti-CD16 antibodies (BD biosciences, San Jose, CA, USA) for 20 min at 4°C. Cells were then fixed with 2% paraformaldehyde and acquired on a FACScanto II cell counter (BD bioscience, San Jose, CA, USA) (200,000 events/sample). To determine the cell frequency in skin biopsies, a punch (4 mm) biopsy was performed in healthy skin and in psoriatic lesions. Tissue biopsies were incubated with Liberase TL (200 μg/ml) (Roche Diagnostics, Germany) for 1 h at 37°C. They were then macerated and filtered with a 40 μm BD (Falcon cell strainer, BD Pharmingen). Cells were stained with anti-MHC II, anti-CD14 and anti-CD16 antibodies (BD biosciences, San Jose, CA, USA), as described above and acquired on a FACScanto II cell counter (BD bioscience, San Jose, CA, USA).

Freshly prepared PBMCs were used. Subpopulations of T cells, B cells, natural killer (NK) cells and antigen-presenting cells (APC) were characterized by surface staining with fluorescence labelled anti-CD3, anti-CD4, anti-CD5, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD38, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1, anti-BDCA2, anti-BDCA3, anti-BDCA4 and anti-slan (Miltenyi Biotec). Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Freshly prepared CSF cells were used directly for FACS analysis. Fluorescence-labeled antibodies for surface staining were used as follows: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1 and anti-slan (Miltenyi Biotec). All cells were measured on a LSR-Fortessa (BD Biosciences) and evaluated by FACS-Diva Software (BD Bioscience).

For immunofluorescent staining, fresh monocytes were incubated with a combination of fluorescein (FITC), phycoerythrin (PE), peridinin chlorophyll protein conjugate (PerCP), and Alexa Fluor-647-labeled monoclonal antibodies (MoAbs). The MoAbs were used in a four-color combination (FITC/PE/PerCP/Alexa Fluor-647): CX3CR1/CD62L/CD14/CD16. Control studies with unstained cells and cells incubated with isotype-matched irrelevant FITC-, PE-, PerCP and Alexa Fluor-647-labeled MoAbs were performed for each experiment. For these procedures, anti-CD62L, anti-CD14 and anti-CD16 were purchased from Becton Dickinson and anti-CX3CR1 purchased from MBL (Naka-ku Nagoya, Japan). Cell acquisition and four-color immunofluorescence analyses were performed using a FACSCalibur flow cytometer (Becton Dickinson) running CellQuest Pro (Becton Dickinson) and FlowJo software (Tree Star Inc, Ashland, Oregon, USA) respectively. In the FSC-SSC dot plot, a biparametric gate was drawn around the monocyte population. This gated population is displayed in a CD14-CD16 dot-plot to define the different monocyte subsets (Additional file 1: Figure S1).