Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue450, CD11b-PE (Tonbo, San Diego, CA), CD11c-APC, CCR5-PE-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7, HLA-DR-FITC, CD3-VioGreen (Miltenyi Biotec), CD3-APC, CD11c-PerCp-Cy5.5, CD1c-PE-dazzle, CD163-APC, HLA-DR-BV570, CD207-APC, CD1a AF700 (Biolegend), CD103-PE-Cy7, CD83-PE, CD14-e780, CD1a-FITC, CD86-e710 (eBiosciences, San Diego, CA), DC-SIGN-FITC, DC-SIGN-PE, DC-SIGN-APC (R&D systems, Minneapolis, MN). Dead cells were excluded with 7AAD (Southern Biotech) or zombie dye yellow staining (Biolegend). Analysis was performed on 8-color MACSQuant 10 (Miltenyi biotech) or Gallios (Beckman Coulter) flow cytometers and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers is shown as percentage of positive cells. Fluorescence minus one (FMO) strategy was used to establish appropriate gates. The gating strategy is shown in supplementary Figure 1 .
Cd83 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD83-PE is a fluorescently-labeled antibody used for the detection and analysis of CD83-expressing cells in flow cytometry applications. CD83 is a member of the immunoglobulin superfamily and is a marker of mature dendritic cells. This product provides a reliable tool for the identification and study of CD83-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Aluminum hydroxide, by Thermo Fisher Scientific (3 mentions)
Cd80 pe, by Thermo Fisher Scientific (3 mentions)
Zombie dye yellow staining, by BioLegend (2 mentions)
Cd45 vioblue450, by Cytek Biosciences (2 mentions)
Cd3 viogreen, by Miltenyi Biotec (2 mentions)
Cd103 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd11c percp cy5, by BioLegend (2 mentions)
Hla dr fitc, by Miltenyi Biotec (2 mentions)
Dc sign fitc, by R&D Systems (2 mentions)
Dc sign pe, by R&D Systems (2 mentions)
Ccr7 pe cy7, by Miltenyi Biotec (2 mentions)
Cd1a af700, by BioLegend (2 mentions)
Cd1a fitc, by Thermo Fisher Scientific (2 mentions)
Cd86 e710, by Thermo Fisher Scientific (2 mentions)
Dc sign apc, by R&D Systems (2 mentions)
Cd163 apc, by BioLegend (2 mentions)
Cd207 apc, by BioLegend (2 mentions)
Cd3 apc, by Miltenyi Biotec (2 mentions)
Ccr5 pe cy7, by BD (2 mentions)
Cd11b pe, by Cytek Biosciences (2 mentions)
8 protocols using cd83 pe
1
Multicolor Flow Cytometry Analysis
2
Flow Cytometric Characterization of BMDCs
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The BMDCs were incubated with mouse Fc block (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°. Fluorochrome‐labelled monoclonal antibodies used in the experiments included anti‐mouse CD80‐allophycocyanin (APC), CD86‐phycoerythrin (PE), MHC‐II‐PE, CD11c‐APC, CD40‐APC, CD83‐PE, CCR7‐PE, CCR5‐APC, CXCR3‐APC, programmed death‐ligand 1 (PD‐L1) ‐PE, CD3e‐FITC, CD4‐PE, CD8‐APC, IL‐17‐PE, IL‐10‐PE, interferon‐γ (IFN‐γ) ‐PE‐Cy7, Foxp3‐APC and CD25‐PE (eBioscience) and their corresponding isotypic controls. For intracellular cytokine staining, cells were pre‐treated with Golgi Stop (BD Biosciences, Franklin Lakes, NJ) and were then stained with fluorochrome‐labelled surface staining monoclonal antibodies or isotypic control for 30 min. A Cytofix Cytoperm kit (BD Biosciences) was then used to permeabilize and fix cells at 4° for another 30 min. Perm wash buffer was used to wash the processed cells before staining them with anti‐IFN‐γ, anti‐IL‐17 and anti‐IL‐10 monoclonal antibodies and isotypic controls for 30 min at 4°. Anti‐mouse Foxp3 staining set APC (eBioscience) was used for Foxp3 immunostaining. Data were captured by flow cytometry (Beckman Coulter, Brea, CA) and analysed using the flowjo software (FlowJo, Ashland, OR).
3
Multicolor Flow Cytometry Analysis
4
Flow Cytometric Immunostaining of DCs
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DCs were stained with specific monoclonal antibodies CD83-PE and HLA-DR-FITC surface markers (eBiosciences, USA). One hundred microliters of cells at 75 × 10 3 /ml were incubated in the dark for 30-45 min with 20 μl of FITC-conjugated antibodies at 4 °C. The cells were washed with cold phosphate buffer saline (PBS), fixed in 1% formaldehyde, and resuspended in 200 μl PBS (Applied Biosystems) [5] .
5
Phenotypic Analysis of CD1a+ Cells
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Cells were stained with monoclonal antibodies directed against: CD1a-PE, HLA-DR-FITC, CD86-APC, (Biolegend, San Diego, CA), CD83-PE (eBioscience, San Diego, CA). Analyses were performed with LSRII flow cytometer (BD Biosciences, Le Pont de Claix, France). All the cells used in this study expressed CD1a (#100%) at their surface.
6
Comprehensive IgE Antibody Detection Assay
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A peroxidase-labeled mouse anti-human IgE Fc antibody and peroxidase-labeled goat anti-mouse IgE, IgG1 and IgG2a Fc antibodies were obtained from SouthernBiotech, USA (9160-05, 1110-05, 1070-05 and 1155-05); tetramethylbenzidine (TMB) was purchased from Solarbio, China (R1200); aluminum hydroxide was obtained from Thermo Fisher, USA (77161); and LPS was purchased from Sigma, USA (L3012). ELISA kits for IL-4, IFN-γ and TNF-α were obtained from Ebioscience, USA (88-7044, 88–7314 and 88–7324); ELISA kits for IL-5 and IL-13 were purchased from 4 A Biotech, China (CME0003, CME0009); an IRF4 antibody was obtained from Cell Signaling, USA (4964); an antibody against GAPDH was procured from Proteintech, China (10494-1-AP); a TLR4 signaling inhibitor was purchased from Invivogen, USA (CLI-095); an anti-mouse TLR2 Ab was obtained from Biolegend, USA (121802); the PE-CD80, PE-CD83, FITC-MHCII and FITC-CD40 antibodies were obtained from Ebioscience, USA (12–0801, 12–0831, 11–5321 and 11–0402); and mouse GM-CSF and IL-4 were purchased from Sino Biological, China (51048-M07H, 51084-M08B). Anti-CD3 and anti-CD28 antibodies were obtained from Ebioscience, USA (16-0031-82, 16-0281-82).
7
Murine Immunology Assays Protocol
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PE-CD80, PE-CD83 and FITC-CD40 antibodies were purchased from Ebioscience, USA (12-0801, 12-0831 and 11-0402). Lipopolysaccharide (LPS) was purchased from Sigma, USA (L3012). Mouse GM-CSF and IL-4 were from Sino Biological, China (51048-M07H, 51084-M08B). Anti-CD3 and anti-CD28 antibodies were obtained from Ebioscience, USA (16-0031-82, 16-0281-82). Aluminum hydroxide was from Thermo Fisher, USA (77161). Peroxidase-labeled goat anti-mouse IgE, IgG1 and IgG2a Fc antibody were from Southernbiotech, USA (1110-05, 1070-05 and 1155-05). ELISA kits for IL-5 and IL-13 detection were from 4A Biotech, China (CME0003, CME0009). ELISA kits for IL-4 and IFN-γ were purchased from Ebioscience, USA (88-7044, 88-7314). Anti-mouse TLR2 antibody and Mouse IgG1, κ Isotype Ctrl were obtained from Biolegend, USA (121802, 400101). TLR4 signaling inhibitor was from Invivogen, USA (CLI-095). DNase I and collagenase D were from Sangon Biotech, China (B002004 and A004186). APC-CD45, FITC-NK-1.1, FITC-CD19 and PE-CD90 were obtained from Biolegend, USA (103111, 108705, 115505 and 205903). PerCP-IL-33R and FITC-Lineage antibodies were purchased from Ebioscience, USA (46-9333, 22-7770). PerCP-CD4, FITC-IL-4, PE-IFN-γ, PE-CD11c and PerCP-Siglec-F were purchased from Ebioscience, USA (46-0041, 11-7042, 12-7311, 12-0114 and 46-1702).
8
Quantifying IgE and IgG1 Antibodies
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The using antibodies for measuring IgE and IgG1 were purchased from Southern biotech, USA. PE-CD80, PE-CD83 and MHC II antibodies were purchased from Ebioscience, USA (12-0801, 12-0831 and 11-5321). Aluminum hydroxide and Thetetramethylbenzidine (TMB) was purchased from Thermo Fisher, USA (77161) and Solarbio, China (R1200), respectively. ELISA kits for IL-4 and IFN-γ detection were purchased from Ebioscience, USA (88-7044 and 88-7314). PerCP-CD4, FITC-IL-4, PE-IFN-γ, PE-CD11c and PerCP-Siglec-F were obtained from Ebioscience, USA (46-0041, 11-7042, 12-7311, 12-0114 and 46-1702). All other chemicals were all purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade unless otherwise stated.
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