Flow cytometry of CD4+ T cells was performed as previously described (7 (link)). The following antibodies were used for T cell phenotyping: CD45 APC-Cy7 (Biolegend), CD4 APC (Biolegend), FoxP3 PE (Thermo Fisher/eBioscience), RORγt PerCP-Cy5.5 (BD Bioscience), and IL-17A-PE (Biolegend), and dead cells were excluded using Zombie NIR (Biolegend). To detect intracellular IL-17A, isolated lymphocytes were first restimulated with 5 ng/mL phorbal 12-myristate 13-acetate and 500 ng/mL ionomycin in the presence of monensin (Biolegend) for 3.5 h. FoxP3 and RORγt were analyzed in unstimulated cells. The Vβ chain repertoire analysis used the following antibodies: Vβ2 AF647 (Biolegend), Vβ3 BrilliantViolet 510 (BD), Vβ5.1/5.2 PE-Cy7 (Biolegend), Vβ6 BrilliantViolet 650 (BD), Vβ7 FITC (Biolegend), Vβ8.1/8.2 APC-Vio770 (Miltenyi), Vβ10b BrilliantViolet 711 (BD), Vβ11 BrilliantViolet 421 (BD), Vβ12 BrilliantViolet 480 (BD), Vβ13 PerCP-eFluor710 (ThermoFisher/eBioscience), Vβ14 biotin (ThermoFisher/eBioscience) with streptavidin Qdot800 (ThermoFisher), Vβ17a BrilliantViolet 605 (BD), CD45 BrilliantViolet 750 (BD), FoxP3 PE (ThermoFisher/eBioscience) RORγt APC (BD), and CD4 BrilliantViolet 570 (Biolegend). T cell phenotyping data and Vβ chain phenotyping data were acquired using an Aurora spectral cytometer (Cytek) and data were analyzed using SpectroFlo and FlowJo 10 software.
Rorγt percp cy5
Manufactured by BD
The RORγt PerCP-Cy5.5 is a fluorescently-conjugated antibody that binds to the retinoic acid-related orphan receptor gamma t (RORγt) protein. It is designed for use in flow cytometry applications to detect and quantify RORγt-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Il 17a pe, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Rorγt apc, by BD (1 mentions)
Aurora spectral cytometer, by Cytek (1 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Monensin, by BioLegend (1 mentions)
Facsverse flow cytometer, by FlowJo (1 mentions)
True nuclear buffer kit, by BioLegend (1 mentions)
Streptavidin bv421, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Dissociation enzyme mix, by Miltenyi Biotec (1 mentions)
Cd8a buv 737, by BD (1 mentions)
Cd25 apccy7, by Thermo Fisher Scientific (1 mentions)
Cd3 alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
3 protocols using rorγt percp cy5
1
Flow Cytometry Analysis of CD4+ T Cells
2
Comprehensive Immune Cell Profiling
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Cells from PP, colon, and spleen were treated with Ultra-LEAF anti-mouse CD16/32 (Clone 93, BioLegend) and subsequently stained with antibodies against surface markers: CD45-A488 (30-F11), CD3ε-BV421 (145-2C11), CD4-APC/Cy7 (GK1.5), CD25-PE/Cy7 (PC61), and CD304 (Neuropilin-1)-PE (3E12) (all from BioLegend). Intracellular staining for transcription factors was performed by fixing and permeabilizing cells using True nuclear buffer kit (BioLegend) and subsequent blocking with normal mouse serum and staining with anti-FOXP3-APC (150D, BioLegend) and RORγt-PerCP-Cy5.5 (Q31-378, BD Biosciences). Intracellular staining of cLP leukocytes was performed using the Foxp3 Staining Buffer Set (eBioscience). T-cell populations were characterized based on forward and side scatter properties and CD45 expression. For B-cell analysis, single cells isolated from PP and spleen were stained using the following antibodies: CD45-A488 (30-F11), B220-APC/Cy7 (RA3-6B2), CD21/CD35-APC (7E9), IgM- PerCP/Cy5.5 (RMM1), IgA-biotin (RMA-1), and BV421-streptavidin (all from BioLegend).
To distinguish live cells LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used. After staining, cells were acquired on an FACSVerse flow cytometer and data analyzed using FlowJo software.
To distinguish live cells LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used. After staining, cells were acquired on an FACSVerse flow cytometer and data analyzed using FlowJo software.
3
Multiparameter Flow Cytometry of Lymph Node T-cells
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Mediastinal lymph nodes were collected and perfused with dissociation enzyme mix (Miltenyi Biotech, Auburn, CA), finely chopped and incubated for 20 minutes at 37 °C. After enzymatic digestion and red blood cell lysis, samples were incubated with an antibody cocktail consisting of: CD4 FITC, CD3 Alexa Fluor 700, CD25 APC/Cy7 (eBioscience, San Diego, CA) and CD8a BUV 737 (BD Biosciences, San Jose, CA). Cells were then permeabilized and incubated with another antibody cocktail consisting of intracellular markers Foxp3 PE, RORγt PerCP/Cy5.5, T-bet Alexa Fluor 647 (BD Biosciences, San Jose, CA) and GATA-3 PE/Cy7 (R&D Systems, Minneapolis, MN). Cells were analyzed by FACS to determine T-cell subsets in the lymph nodes.
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