Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103–BV711 (Biolegend), CD4-PE-Cy5.5, CD103–PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience). Analysis was performed on BioRad ZE5 flow cytometers (BioRad) using Everest software or Gallios (Beckman Coulter) using Kaluza software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells.
Cd103 pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD103-PE-Cy7 is a fluorescently labeled antibody designed for the detection and analysis of CD103-expressing cells in flow cytometry applications. It contains the PE-Cy7 fluorescent label.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 vioblue450, by Cytek Biosciences (6 mentions)
Cd3 viogreen, by Miltenyi Biotec (6 mentions)
Cd8 fitc, by Cytek Biosciences (4 mentions)
Zombie dye yellow staining, by BioLegend (4 mentions)
Hla dr fitc, by Miltenyi Biotec (4 mentions)
Cd11c percp cy5, by BioLegend (4 mentions)
Cd4 pe, by Thermo Fisher Scientific (3 mentions)
Ccr7 pe cy7, by Miltenyi Biotec (3 mentions)
Macsquant 10, by Miltenyi Biotec (3 mentions)
Cd103 bv711, by BioLegend (2 mentions)
Cd4 pe cy5, by Thermo Fisher Scientific (2 mentions)
Cd8 buv395, by BD (2 mentions)
Cd45 af700, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (2 mentions)
Cd4 apc cy7, by BioLegend (2 mentions)
Cd19 apc, by Cytek Biosciences (2 mentions)
Cd103 pe, by Miltenyi Biotec (2 mentions)
Cd56 apc, by BD (2 mentions)
Dc sign fitc, by R&D Systems (2 mentions)
Dc sign pe, by R&D Systems (2 mentions)
7 protocols using cd103 pe cy7
1
Multiparameter Flow Cytometry Analysis
2
Multi-parameter Flow Cytometry Immunophenotyping
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Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue450, CD8-FITC, CD19-APC (Tonbo, San Diego, CA), HLA-DR-FITC, CD3-viogreen, CD103-PE (Miltenyi Biotec, Auburn, CA), CD11c-PerCp-Cy5.5, CD103-PE-Cy7, CD4-PE (eBioscience, San Diego, CA), CD56-APC (BD Pharmingen, San Diego, CA), CD69-BrilliantViolet510 (BioLegend, San Diego, CA). Dead cells were excluded with 7AAD (Southern Biotech, Birmingham, AL) or zombie dye yellow staining (BioLegend, San Diego, CA). For spectral flow cytometry, the following antibodies were used: CX3CR1-PE eFluor610 (Thermo Fisher, Waltham, MA), CD3-viogreen (Miltenyi Biotec), CD4-BrilliantUV805, CD10-BrilliantViolet650, CCR5-PE-Cy5, CD45-BrilliantUV395 (BD Biosciences, Franklin Lakes, NJ), HLA-DR-AlexaFluor700, CCR7-BrilliantViolet750, CD8-SparkBlue550, CD62L-BrilliantViolet605 (BioLegend). Analysis was performed on Gallios (Beckman Coulter, Brea, CA) or Cytek Aurora 5 lasers configuration (Cytek, Fremont, CA) flow cytometers and data analyzed with FlowJo ( Tree Star, Inc., Ashland, OR) or OMIQ software (www.omiq.ai ). Expression of surface markers is shown as percentage of positive cells. Fluorescence minus one (FMO) strategy was used to establish appropriate gates.
3
Flow Cytometry Analysis of Immune Cell Subsets
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Cell surface markers from the mixed cell suspensions were stained with various combinations of antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103-BV711 (BioLegend), CD4-PE-Cy5.5, CD103-PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience). Cell surface marker expression was measured by the percentage of positive cells analyzed using BioRad ZE5 flow cytometers (BioRad) with Everest software. Data was analyzed with FlowJo Software v.10 (Tree Star Inc., Ashland, OR, www.flowjo.com ). The authors acknowledge the Immune Monitoring and Flow Cytometry Resource at the Norris Cotton Cancer Center at Dartmouth with NCI Cancer Center Support Grant (5P30 CA023108-41) and COBRE Grant (P30GM103415-15) from the National Institute of General Medical Sciences.
4
Multicolor Flow Cytometry Analysis
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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 .
5
Multicolor Flow Cytometry Protocol
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Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45‐vioblue 450, CD8‐FITC, CD19‐APC (Tonbo, San Diego, CA), HLA‐DR‐FITC, CD3‐viogreen, CD103‐PE (Miltenyi Biotec, Auburn, CA, USA), CD11c‐PerCp‐Cy5.5, CD69‐PE‐Cy7 (Biolegend, San Diego, CA), CD103‐PE‐Cy7, CD4‐PE (eBiosciences, San Diego, CA, USA), CD56‐APC (BD Pharmingen, San Diego, CA, USA). 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 were analyzed with FlowJo software (Tree Star, Inc. Ashland, OR, USA). Expression of surface markers is shown as percentage of positive cells and MFI. Fluorescence minus one (FMO) strategy was used to establish appropriate gates. Comparisons between FMO and isotype controls showed no differences. For tissue dendritic cell (DC) quantification, DCs were identified using flow cytometry as CD45+, CD3‐, CD19‐, CD56‐, HLA‐DRhigh, CD11c+ cells as described before (Rodriguez‐Garcia et al., 2017 ), and the cell number was normalized to tissue weight.
6
Multicolor Flow Cytometry Analysis
7
Naive T Cell Proliferation Assay
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Naïve T cells were purified from cryopreserved peripheral blood mononuclear cells (PBMCs) using the Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). After purification, isolated naïve T cells were >99% CCR7+ CD45A+ as determined by flow cytometry with CD3‐APC‐Cy7 (Tonbo), CCR7‐PE‐Cy7 (Miltenyi Biotec), and CD45RA‐APC (Biolegend). Naïve T cells were stained with Cell Proliferation Dye eFluor‐670 (eBioscience) as recommended by the manufacturer. Purified mucosal CD1a+ or CD14+ cells (5 × 103 cells) were plated with naïve T cells (7.5 × 104 cells) (1:15 ratio) in round‐bottom 96‐well plates, in Xvivo 15 media (Invitrogen) supplemented with 10% human AB serum (Valley Biomedical). After 6 days in culture, proliferation of T cells was assessed by flow cytometry after staining with zombie yellow dye (Biolegend) and CD3‐APC‐Cy7, CD8‐FITC (Tonbo), CD4‐PE, CD103‐PE‐Cy7 (eBiosciences), and CD11c‐PerCp‐Cy5.5 (Biolegend). Naïve T cells alone were used as a negative control. For some experiments, TGFβ receptor 1 blocker, SB431542 (10 μmol/L, Tocris Cookson Inc) (Ochiel, Ochsenbauer, et al., 2010 ), was added to the cultures at the beginning of each experiment.
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