Dectin-1 (C-type lectin domain family 7 member A, Dendritic cell-associated C-type lectin 1, DC-associated C-type lectin 1, Beta-glucan receptor, C-type lectin superfamily member 12, CLEC7A, BGR, CLECSF12, DECTIN1, UNQ539/PRO1082) (PE)

Designed to quantitatively determine the percentage of cells bearing Dectin-1/CLEC7A within a population and qualitatively determine the density of Dectin-1/CLEC7A on cell surfaces by flow cytometry. Dectin-1, also known as CLEC7A and the b-glucan receptor, is a 33kD type II transmembrane C-type lectin that participates in the innate immune response to fungal pathogens. Although Dectin-1 structurally resembles other CLEC molecules, it binds its ligands in a calcium-independent manner.1, 2 Mature human Dectin-1 consists of a short N-terminal ITAM-containing cytoplasmic tail, a transmembrane segment, and a C-terminal stalk with carbohydrate recognition domain (CRD) in the extracellular domain.3, 4 Alternate splicing generates one major splice form that lacks the stalk region.3-5 This isoform is expressed on the surface of monocytes, macrophages, myeloid dendritic cells, neutrophils, eosinophils, B cells, and CD4+ T cells.6 The CRD selectively binds b-glucan polymers, a major component of yeast and mycobacterial cell walls.5-7 Yeast b-glucan is accessible to Dectin-1 only during the process of cell budding. Dectin-1 does not recognize the filamentous form of yeast.8 Dectin-1 mediates the phagocytosis of zymosan particles and intact yeast.8-10 In the membrane, Dectin-1 co-localizes with TLR2 in the presence of zymosan, and the two receptors cooperate in ligand recognition and the propagation of proinflammatory signaling.9, 11-13 Dectin-1 also interacts with tetraspanin CD37. This increases its stability on the cell membrane and inhibits ligand-induced signaling.14 Dectin-1 knockout mice show increased susceptibility to pathogenic infection.15-16 The CRD of human Dectin-1 shares 77%, 60%, and 60% amino acid (aa) sequence identity with that of bovine, mouse and rat Dectin-1, respectively. It shares 29%-39% aa sequence identity with the CRD of other subgroup members, including CLEC-1, CLEC-2, CLEC9A, CLEC12B, LOX-1, and MICL. Principle: Washed cells are incubated with the phycoerythrin-labeled monoclonal antibody, which binds to cells expressing Dectin-1/CLEC7A. Unbound phycoerythrin-conjugated antibody is then washed from the cells. Cells expressing Dectin-1/CLEC7A are fluorescently stained, with the intensity of staining directly proportional to the density of expression of Dectin-1/CLEC7A. Cell surface expression of Dectin-1/CLEC7A is determined by flow cytometric analysis using 488 nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 565-605 nm. Reagent Preparation: Phycoerythrin-conjugated mouse anti-human Dectin-1/CLEC7A: Use as is; no preparation necessary. Human monocytes were stained with PE-conjugated anti-human Dectin-1/CLEC7A filled histogram) or isotype control (open histogram). Sample Preparation Peripheral blood cells: Whole blood should be collected in evacuated tubes containing EDTA or heparin as the anticoagulant. Contaminating serum components should be removed by washing the cells three times in an isotonic phosphate buffer (supplemented with 0.5% BSA) by centrifugation at 500 x g for 5 minutes. Transfer 50il of packed cells to a 5ml tube for staining with the monoclonal antibody. Whole blood will require lysis of RBC following the staining procedure. Cell Cultures: Continuous cell lines or activated cell cultures should be centrifuged at 500 x g for 5 minutes and washed three times in an isotonic PBS buffer (supplemented with 0.5% BSA), as described above, to remove any residual growth factors that may be present in the culture medium. Cells should then be resuspended in the same buffer to a final concentration of 4 x 106 cells/ml and 25il of cells (1x10e5) transferred to a 5 ml tube for staining. Note: Adherent cell lines may require pretreatment with 0.5 mM EDTA to facilitate removal from substrate. Cells that require trypsinization to enable removal from substrate should be further incubated in medium for 6-10 hours on a rocker platform to enable regeneration of the receptors. The use of the rocker platform will prevent reattachment to the substrate. Sample Staining: 1) Cells should be Fc-blocked by treatment with 1ug of human IgG/105 cells for 15 minutes at room temperature prior to staining. Do not wash excess blocking IgG from this reaction. 2) Transfer 25il of the Fc-blocked cells (1 x 10e5 cells) or 50il of packed whole blood to a 5 ml tube. 3) Add 10il of PE-conjugated Dectin-1/CLEC7A reagent. 4) Incubate for 30-45 minutes at 2 degrees -8 degrees C. 5) Following this incubation, remove unreacted Dectin-1/CLEC7A reagent by washing the cells twice in 4 ml of the same PBS buffer (note: whole blood will require an RBC lysis step at this point using any commercially available lysing reagent, such as a Whole Blood Lysing Kit). 6) Finally, resuspend the cells in 200-400il of PBS buffer for final flow cytometric analysis. 7) As a control for analysis, cells in a separate tube should be treated with PE-labeled mouse IgG2B antibody. This procedure may need modification, depending upon final utilization. Storage: Reagents are stable for twelve months from date of receipt when stored in the dark at 2-8 degrees C.