Pe anti cd14 clone hcd14

For dual and three-colour staining, the following monoclonal antibodies were used: Phycoerythrin-conjugated anti-CD56 (NKH-1), Phycoerythrin-Texas Red-conjugated anti-CD3 (UCHT1) and anti-NKG2A unconjugated (Z199), all purchased from Beckman Coulter (Fullerton, California), anti-NKG2C unconjugated (clone 134522; R&D), PE-anti-CD14 (clone HCD14; BioLegend) and fluorescein isothiocyanate (FITC)-conjugated goat F(ab')₂ antimouse (GAM) IgG (F0479; Dako). For the detection of HLA-E surface molecules, three different unconjugated monoclonal antibodies were tested: MEM-E/06, MEM-E/07 and MEM-E/08 (kindly provided by Vaclav Horejsi), recognising surface HLA-E molecules, whose HLA-E specificity was previously defined by flow cytometry on the Third International Conference on HLA-G (Paris, July 2003).26 27 (link) According to the literature, MEM-E/08 resulted as the better one in terms of specificity and was selected for further experiments. The surface expression of HLA-B27 was also analysed (by means of clone ME1) to exclude cross-reactivity of MEM-E/08 with HLA-B27. The expression of the two antigens on cell surface did not overlap. In order to avoid possible unspecific staining, as described in table 1 in Lo Monaco et al,27 (link)
²⁷ patients and controls were not HLA-A24-positive, HLA-B7-positive, HLA-B54-positive or HLA-B65-positive.

Emigrated skin DC were collected 4 days after the start of skin explant cultures. Cells were permeabilized and stained with the BD-Fixation/Perm kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instruction. Targeting mAbs against Langerin and DEC-205 were detected with anti-mouse-Ig-APC (BioLegend), followed by blocking with 100 μg/ml mouse gamma globulin for 5 min (Jackson ImmunoResearch Laboratories) and staining of DC with anti-CD1a-FITC (clone HI149, BD Biosciences), anti-CD163-PerCP-Cy5.5 (clone GHI/61, BioLegend), anti-CD14-PE (clone HCD14, BioLegend), anti-HLA-DR-PE-Cy7 (clone L243, BioLegend) for 10 min at 4°C. Appropriate isotype controls from Biolegend were used for FACS stainings to confirm specific staining. Analyses were performed on a FACS Calibur and Canto II instrument (BD Biosciences). The percentage of targeted skin DC was determined by pregating on viable HLA-DR⁺ cells, followed by gating for the various skin DC subsets with CD1a and CD14. In the three different skin DC subsets, cells positive for the targeting mAb were determined by setting the gate on DC from skin explants injected with isotype controls.