For cytofluorimetric analysis, cells were stained with surface antibodies in PBS 5% FCS for 20 min at 4 °C. The following antibodies were used: CD133-APC, EpCAM -VioBlue, CD105-PE, CD90-VioBlue, IFNγ-PE, CD107a-APC, CCR7-FITC, CD163-PerCP-Vio700, CD206-VioBlue, HLA-DR-PerCP, CD80-APC (MIltenyi Biotec); CD56-PC7, NKp30-PE (Beckman Coulter); CD29-PE (ImmunoTools); CD146-PC7, CD73-FITC, NKp46-V450 (BD Biosciences); E-cadherin-APC (Thermo Fisher Scientific); N-cadherin-PE (Abcam); DNAM1-PE (Biolegend).
Dnam 1 pe
Manufactured by BioLegend
Sourced in United States
DNAM-1-PE is a fluorochrome-conjugated monoclonal antibody that binds to the DNAM-1 (CD226) protein expressed on the surface of various immune cells. DNAM-1 is an adhesion molecule involved in cell-cell interactions and immune cell activation. The PE (Phycoerythrin) fluorochrome allows for the detection and analysis of DNAM-1-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Nkp46 pe cyanine7, by BioLegend (2 mentions)
Cd105 pe, by Miltenyi Biotec (1 mentions)
Cd80 apc, by Miltenyi Biotec (1 mentions)
Cd107a apc, by Miltenyi Biotec (1 mentions)
Ifnγ pe, by Miltenyi Biotec (1 mentions)
Epcam vioblue, by Miltenyi Biotec (1 mentions)
Ccr7 fitc, by Miltenyi Biotec (1 mentions)
Hla dr percp, by Miltenyi Biotec (1 mentions)
E cadherin apc, by Thermo Fisher Scientific (1 mentions)
N cadherin pe, by Abcam (1 mentions)
Cd133 apc, by Miltenyi Biotec (1 mentions)
Cd56 pe, by BioLegend (1 mentions)
Cd16 pe, by BioLegend (1 mentions)
Dnam 1 fitc, by BioLegend (1 mentions)
Antimouse cd3 percp cyanine 5, by BioLegend (1 mentions)
Fitc nkp46, by BioLegend (1 mentions)
Nkg2d apc, by BioLegend (1 mentions)
Pe isotype control, by BioLegend (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Tigit bv421, by BioLegend (1 mentions)
4 protocols using dnam 1 pe
1
Multi-Marker Cytometry Profiling
2
NK Cell Phenotyping in Mouse and Human
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After cocultivating NK cells and PC cells with different treatments for three days, NK cells were collected and washed twice with PBS, and then, anti-human CD3-PerCP/Cyanine5.5, CD16-PE, CD56-PE, NKG2D-APC, NKp46-PE/Cyanine7, and DNAM-1-FITC antibodies were added (BioLegend, San Diego, CA, USA) according to the instructions. The cells were incubated for 20 min in the dark and washed twice with PBS. For the detection of NK cells in mouse peripheral blood, 100 μl mouse peripheral blood was collected, and anti-mouse CD3-PerCP/Cyanine5.5, NK1.1-PE-Cyanine7, NKG2D-APC, NKp46-FITC, and DNAM-1-PE antibodies were added (BioLegend, San Diego, CA, USA) and incubated at room temperature for 20 minutes in the dark. Then, 2 ml RBC lysis solution was added to each tube, mixed well, and incubated for 15 min. Then, the samples were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. All samples were then analyzed by multicolor flow cytometry (BD, USA).
3
Flow Cytometric Analysis of T-Cell Markers
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Cell surface staining for flow cytometry was performed using the antibodies CD3-APC-Cy7 (Clone: SK7), γδ-PE-Cy7 (Clone: B1), TIGIT-BV421 (Clone: A15153G), and DNAM-1-PE (Clone: 11Aδ) together with a BV421 isotype Control (Clone: G155-178) and a PE isotype Control (Biolegend, San Diego, USA). The Foxp3-Alexaflour 647 (Clone: 236A/E7) fluorescent antibody was stained independently.
4
Evaluating NK Cell Response to Olaparib
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Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by density gradient centrifugation and resuspended in RPMI supplemented with 10% FBS and 1% penicillin–streptomycin (complete RPMI (cRPMI), Gibco). PBMC were seeded at a density of 500,000 cells per 500 µL of cRPMI and treated for 24 h with 5 μM or 10 μM of Olaparib. Following treatment, the cells were surface stained for flow cytometric analysis using CD56-FITC (BioLegend), CD3-APC-Cyanine7 (BioLegend), and the following antibodies for activating NKRs, NKp46-PE-Cyanine7 (BioLegend), DNAM-1-PE (BioLegend), NKp30-BV421 (BioLegend), NKG2D-PE-Cyanine5 (BioLegend), CD16-PE-Cyanine7 (BioLegend), inhibitory receptors NKG2A-APC (Miltenyi Biotec), PD-1-PE-Cyanine7 (BioLegend), and TIGIT-PerCP-Cyanine5.5 (BioLegend), death receptor ligands TRAIL-APC (BioLegend) and FasL-BV421 (BioLegend), and phenotypic markers, CD57-PE (BioLegend), CD69-BV510 (BioLegend), and CD27-Pacific blue (BioLegend). All samples were acquired using the BD FACS CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo v10 software (BD Biosciences). NK cells were defined as CD56+ CD3− in the lymphocyte gate. The gating strategies are available in Supplementary Figure S1 .
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