The generation of the dectin-1-knockout mice is described in the Supplementary Methods online. Mice were maintained in accordance with institutional guidelines at the University of Oxford, London (Imperial College) and the University of Cape Town. Clec7a+/+ and Clec7a−/− mice were on a mixed 129/Sv × C57BL/6 genetic background unless stated otherwise.
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Dectin 1
Dectin 1
Dectin-1 is a C-type lectin receptor that plays a key role in the recognition and response to fungal pathogens.
It is expresed on the surface of myeloid cells, such as macrophages and dendritic cells, and binds to beta-glucans found in fungal cell walls.
Dectin-1 activation triggers intracellular signaling pathways that promote phagocytosis, respiratory burst, and the production of proinflammatory cytokines, chemokines, and eicosanoids, contributing to antifunal immunity.
Dysregulation of Dectin-1 function has been implicated in the pathogenesis of fungal infections, allergic diseases, and autoimmune disorders.
Understanding the molecular mechanisms underlying Dectin-1 signaling and its interactions with fungal pathogens is an area of active research with therapeutic potential.
It is expresed on the surface of myeloid cells, such as macrophages and dendritic cells, and binds to beta-glucans found in fungal cell walls.
Dectin-1 activation triggers intracellular signaling pathways that promote phagocytosis, respiratory burst, and the production of proinflammatory cytokines, chemokines, and eicosanoids, contributing to antifunal immunity.
Dysregulation of Dectin-1 function has been implicated in the pathogenesis of fungal infections, allergic diseases, and autoimmune disorders.
Understanding the molecular mechanisms underlying Dectin-1 signaling and its interactions with fungal pathogens is an area of active research with therapeutic potential.
Most cited protocols related to «Dectin 1»
dectin 1
Genetic Background
Mice, House
Mice, Knockout
Cells
Common Cold
Cytokine
Edetic Acid
Flow Cytometry
Inflammation
Leukocytes
Mus
Peritoneal Fluid
Population Group
Zymosan
Cell wall mannan, β-glucan and chitin contents were determined by hydrolysis of these oligosaccharides and quantification by high-performance anion-exchange chromatography, as described previously [47] (link). To detect chitin, ex vivo isolated C. albicans cells were stained and quantified using Calcofluor White, as previously described [22] (link). TEM analysis was performed as previously described [36] (link).
To detect exposed β-glucan, C57BL/6J mice were injected in the tail vein with 5.2×104 CFU of either SC5314-GFP or ATCC18804-GFP. SC5314-GFP and ATCC18804-GFP strains were created by transformation with the pENO1-yEGFP3-NAT plasmid and verified by PCR as described previously [17] (link). After nine days, mice were sacrificed and the kidneys were harvested, homogenized, and processed as described [17] (link). Homogenates were stained with anti-β-glucan antibody (Biosupplies, Inc., Australia) at a concentration of 1.7 µg/ml, then stained with goat anti-mouse Cy3 antibody (Jackson Immunoresearch) at a concentration of 3.8 µg/ml. For soluble Dectin-1-Fc staining, homogenates were instead stained with Alexa647-labelled Dectin-1-Fc [48] (link) at a concentration of 17 µg/ml and then with donkey anti-human IgG Cy3 antibody (Jackson Immunoresearch) at a concentration of 0.8 µg/ml. Cells were visualized by optical sectioning fluorescence microscopy using a Zeiss Axiovision Vivotome microscope (Carl Zeiss Microscopy, LLC). Live cells were identified based on characteristic EGFP fluorescence. Maximum projection images were quantified using Cellprofiler (www.cellprofiler.org ) as described [17] (link). Briefly, EGFP fluorescence was used to manually define individual cell segments and average fluorescence intensity of β-glucan or Dectin-1-CRD fluorescence was measured for the whole cell segment. Cells labelled without primary antibody or Dectin-1-CRD were used as negative controls. In vitro grown cells were stained with soluble Dectin-1 at 5 µg/ml and then with anti-human IgG antibody (used at 1∶200) (Jackson Immunoresearch). Controls were stained with secondary antibody only.
To detect exposed β-glucan, C57BL/6J mice were injected in the tail vein with 5.2×104 CFU of either SC5314-GFP or ATCC18804-GFP. SC5314-GFP and ATCC18804-GFP strains were created by transformation with the pENO1-yEGFP3-NAT plasmid and verified by PCR as described previously [17] (link). After nine days, mice were sacrificed and the kidneys were harvested, homogenized, and processed as described [17] (link). Homogenates were stained with anti-β-glucan antibody (Biosupplies, Inc., Australia) at a concentration of 1.7 µg/ml, then stained with goat anti-mouse Cy3 antibody (Jackson Immunoresearch) at a concentration of 3.8 µg/ml. For soluble Dectin-1-Fc staining, homogenates were instead stained with Alexa647-labelled Dectin-1-Fc [48] (link) at a concentration of 17 µg/ml and then with donkey anti-human IgG Cy3 antibody (Jackson Immunoresearch) at a concentration of 0.8 µg/ml. Cells were visualized by optical sectioning fluorescence microscopy using a Zeiss Axiovision Vivotome microscope (Carl Zeiss Microscopy, LLC). Live cells were identified based on characteristic EGFP fluorescence. Maximum projection images were quantified using Cellprofiler (
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Alexa Fluor 647
Anions
anti-IgG
Antibodies, Anti-Idiotypic
AT 17
beta-Glucans
calcofluor white
Candida albicans
Cells
Cell Wall
Chitin
Chromatography
dectin 1
Equus asinus
Fluorescence
Goat
Homo sapiens
Hydrolysis
Immunoglobulins
Kidney
Mannans
Mice, Inbred C57BL
Microscopy
Microscopy, Fluorescence
Mus
Oligosaccharides
Plasmids
Strains
Tail
Veins
anti-IgG
Antibodies, Anti-Idiotypic
beta-Glucans
Cells
dectin 1
Enzyme-Linked Immunosorbent Assay
Escherichia coli
IL10 protein, human
laminaran
Mannans
Monocytes
Mycobacterium tuberculosis
N-palmitoyl-S-(2,3-bis(palmitoyloxy)propyl)cysteinyl-seryl-lysyl-lysyl-lysyl-lysine
Pargyline
Psychological Inhibition
Raf1 protein, human
Tumor Necrosis Factor-alpha
Most recents protocols related to «Dectin 1»
Expression of Dectin-1 was knocked down in vitro, among murine BMDCs by post transcriptional gene silencing (PTGS) using RNA interference (RNAi). An siRNA molecule was constructed using the Ambion Silencer® siRNA Construction Kit (Life Technologies, USA). A pair of 29-mer ssDNA-oligonucleotides was taken for the synthesis of each siRNA strand (Supplementary Table 6 ). Individual strands of the siRNA were then transcribed, using the T7 promoter, hybridized and further cleaved, as described in the manufacturer’s protocol. Accordingly, a double stranded 21-mer siRNA with 3’ Uridine-dimers as overhangs in both strands was synthesized for Dectin-1 knockdown (5’-GGCCCAGGGGAUCAGAGAAUU-3’ as the guide strand and 5’-UUCUCUGAUCCCCUGGGCCUU-3’ as the passenger strand). Following quantification by Nanodrop Spectrophotometer (MicroDigital Co., Ltd. Korea), this siRNA was introduced within mBMDCs by transfection, using the Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific, MA, USA) and serum-reduced Opti-MEM® medium (Thermo Fisher Scientific, MA, USA). The siRNA (50 µM) and Lipofectamine (6 µl) were separately added to two Opti-MEM aliquots (250 µl each) and both were kept at room temperature for 5 min. Then, both were mixed, up to a final volume of 500 µl and kept at room temperature for 20 min. This mixture was added in vitro to 2-h serum-deprived, 70% confluent culture of mBMDCs, so that the final concentration of the siRNA becomes 50 nM within the media. A non-specific siRNA was parallelly used as control. Expression of Dectin-1 was quantified in different groups of cells by western botting, to check the extent of downregulation. The mBMDCs with suppressed levels of Dectin-1 were further checked by microscopy, PCR, flow-cytometry and ELISA for NLGP-binding and consequent cytokine-release patterns, compared to controls.
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The extended β-Glucan chains present on the surface of the ligands of Dectin-1 (CLEC7A) receptors, adhere to these receptors to bring about the overall receptor-ligand binding. Thus, in an attempt to understand the molecular mechanisms of NLGP-Dectin-1 binding in the best possible way, favorable associations of Dectin-1 and β-Glucans were elucidated in terms of thermodynamic stability in silico, by molecular docking studies. Two different β-Glucan chains (CHEBI:133,802 and CHEBI:18,246) were procured from the online dictionary ‘Chemical Entities of Biological Interest’ (ChEBI) of EMBL-EBI as Molfiles (.mol), which were converted to the partially charged file format (.pdbqt) by Open Babel GUI (Open Babel: The Open Source Chemistry Toolbox). Three common β-Glucan structures namely, β-D-Glc-(1 → 3)-[β-D-Glc-(1 → 6)]-β-D-Glc-(1 → 3)-β-D-Glc, β-D-Glc-(1 → 3)-β-D-Glc-(1 → 3)-β-D-Glc-(1 → 3)-β-D-Glc and β-D-Glc-(1 → 4)-β-D-Glc-(1 → 4)-β-D-Glc were prepared as small molecule-ligands from these two structures in BIOVIA Discovery Studio Visualizer (Dassault Systemes, Île-de-France, France). The quaternary dimeric crystal structure of murine Dectin-1 (2BPE) was obtained from RCSB Protein Data Bank. This crystal structure of Dectin-1 was refined by removing the embedded water molecules and the excess hetero atoms, followed by repairing the missing atoms of the amino acid residues and polar hydrogens and addition and uniform distribution of Kollman charges, in AutoDock 4.2.6 (Scripps Research, La Jolla, CA, USA).
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Six glycoprotein receptors present on mBMDC surfaces, namely, Mannose binding receptor, Dectin-1, Dectin-2, DC-SIGN, DEC-205 and DNGR1 were neutralized. Mannan (10 µM) (Sigma-Aldrich, St. Louis, Missouri, USA), obtained from Saccharomyces cerevisiae and Laminarin (10 µM) (InvivoGen, San Diego, CA, USA), prepared from Laminaria digitata were used to block MBRs and Dectin-1 receptors respectively. Neutralizing antibodies (5 µg/ml) were used to block the other four receptor types. After addition of the corresponding inhibitors, each of the groups of cells was incubated within complete RPMI-1640 medium, for one hr at 37 °C, supplied with 5% CO2. The receptor-neutralizations were done prior to the addition of NLGP or NLGP-FITC to the mBMDCs.
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Specific pathogen-free (SPF) male C57BL/6 and BALB/c mice aged 5–7 weeks old were purchased from Charles River Laboratories Japan. Dectin-2- and Dectin-1-deficient mice in the C57BL/6 background were described previously [28 (link),60 (link)]. Mincle- and MCL-deficient mice in the C57BL/6 background were described previously [61 (link),62 (link)]. Myd88-deficient mice in the C57BL/6 background [63 (link)] were purchased from Oriental Bioservice (Kyoto, Japan). Card9-deficient mice in the C57BL/6 background were described previously [64 (link)]. All mice were housed under SPF conditions with food and water available ad libitum.
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Centre of the active site of Dectin-1 was determined from the X, Y and Z coordinates of the active site amino acids, obtained from the.pdb file of the crystal structure. A spatial grid-box was generated using Autogrid 4.2.6 around the centre, taking 80 points in each dimension with 0.5 Å spacing. Dockings were performed according to the Genetic algorithm with 30 runs for each ligand in AutoDock 4.2.6 (Scripps Research, La Jolla, CA, USA). The Lamarckian GA (4.2) outcomes of docked complexes were analyzed and the ones with the lowest binding free energy values were taken (with.pdbqt extensions) as the most stable conformations for each ligand-receptor docked complex. The individual root mean square deviations (RMSDs), binding energies (Kcal/mol) and inhibition constants (Ki) for each ligand (with Dectin-1) were found in the respective Docking log files and the numbers of generated H-bonds along with the amino acids of Dectin-1, involved in each interaction was obtained using BIOVIA Discovery Studio Visualizer (Dassault Systemes, Île-de-France, France). All diagrams, representing the molecular interactions were prepared in BIOVIA Discovery Studio Visualizer.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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Laminarin is a polysaccharide compound produced by certain types of brown algae. It is commonly used in laboratory settings as a reference standard and calibration material for various analytical and biomedical applications. Laminarin exhibits a linear structure composed of glucose monomers linked together.
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Curdlan is a lab equipment product manufactured by Merck Group. It is a naturally occurring polysaccharide that functions as a gelling agent and thickener in various applications.
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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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C57BL/6 is a widely used inbred mouse strain. It is a robust, readily available laboratory mouse model.
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The FACSCanto II is a flow cytometer instrument designed for multi-parameter analysis of single cells. It features a solid-state diode laser and up to four fluorescence detectors for simultaneous measurement of multiple cellular parameters.
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Zymosan is a cell wall preparation derived from Saccharomyces cerevisiae (baker's yeast). It is composed of a complex mixture of polysaccharides, including β-glucans and mannans. Zymosan is commonly used as a tool for stimulating innate immune responses in vitro and in vivo.
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Streptomycin is a broad-spectrum antibiotic used in laboratory settings. It functions as a protein synthesis inhibitor, targeting the 30S subunit of bacterial ribosomes, which plays a crucial role in the translation of genetic information into proteins. Streptomycin is commonly used in microbiological research and applications that require selective inhibition of bacterial growth.
More about "Dectin 1"
Dectin-1, also known as C-type lectin domain family 7 member A (CLEC7A), is a crucial pattern recognition receptor (PRR) that plays a pivotal role in the detection and response to fungal pathogens.
This transmembrane receptor is primarily expressed on the surface of myeloid cells, such as macrophages, dendritic cells, and neutrophils.
Dectin-1 recognizes and binds to beta-glucans, a key component of fungal cell walls.
This interaction triggers a cascade of intracellular signaling pathways that promote phagocytosis, respiratory burst, and the production of pro-inflammatory cytokines, chemokines, and eicosanoids.
These immune responses contribute to the host's antifungal defenses.
Dysregulation of Dectin-1 function has been implicated in the pathogenesis of various fungal infections, allergic diseases, and autoimmune disorders.
Understanding the molecular mechanisms underlying Dectin-1 signaling and its interactions with fungal pathogens is an area of active research with significant therapeutic potential.
Researchers commonly utilize a variety of experimental tools and techniques to study Dectin-1, including FBS (Fetal Bovine Serum) for cell culture, TRIzol reagent for RNA extraction, Laminarin and Curdlan as Dectin-1 agonists, FACSCalibur and FACSCanto II flow cytometers for immune cell analysis, and the RNeasy Mini Kit for RNA purification.
Additionally, the C57BL/6 mouse strain is often employed as a model organism for in vivo studies.
Zymosan, a fungal cell wall component, and Streptomycin, an antibiotic, are also frequently used in Dectin-1 research.
This transmembrane receptor is primarily expressed on the surface of myeloid cells, such as macrophages, dendritic cells, and neutrophils.
Dectin-1 recognizes and binds to beta-glucans, a key component of fungal cell walls.
This interaction triggers a cascade of intracellular signaling pathways that promote phagocytosis, respiratory burst, and the production of pro-inflammatory cytokines, chemokines, and eicosanoids.
These immune responses contribute to the host's antifungal defenses.
Dysregulation of Dectin-1 function has been implicated in the pathogenesis of various fungal infections, allergic diseases, and autoimmune disorders.
Understanding the molecular mechanisms underlying Dectin-1 signaling and its interactions with fungal pathogens is an area of active research with significant therapeutic potential.
Researchers commonly utilize a variety of experimental tools and techniques to study Dectin-1, including FBS (Fetal Bovine Serum) for cell culture, TRIzol reagent for RNA extraction, Laminarin and Curdlan as Dectin-1 agonists, FACSCalibur and FACSCanto II flow cytometers for immune cell analysis, and the RNeasy Mini Kit for RNA purification.
Additionally, the C57BL/6 mouse strain is often employed as a model organism for in vivo studies.
Zymosan, a fungal cell wall component, and Streptomycin, an antibiotic, are also frequently used in Dectin-1 research.