The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Rorγt pe
Manufactured by BD
RORγt-PE is a fluorochrome-conjugated monoclonal antibody that specifically binds to the retinoic acid-related orphan receptor gamma t (RORγt) protein. RORγt is a transcription factor that is essential for the differentiation of T helper 17 (Th17) cells. The RORγt-PE antibody can be used to detect and quantify RORγt-expressing cells, such as Th17 cells, in flow cytometry applications.
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Lab products found in correlation
Foxp3 fitc, by BD (2 mentions)
T bet pe, by BD (2 mentions)
Cd278 icos biotin, by BD (2 mentions)
Cd25 bb515, by BD (2 mentions)
Live dead fixable near ir stain, by Thermo Fisher Scientific (2 mentions)
Cd44 pe cy7, by BD (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Canto 2, by BD (2 mentions)
Cxcr3 apc, by BD (1 mentions)
Ifn γ pe, by BD (1 mentions)
Cd27 pe cy7, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Gata3 pe, by BD (1 mentions)
Cd4 a700, by BD (1 mentions)
Cd24 pe cf594, by BD (1 mentions)
Il 17 percpcy5, by BD (1 mentions)
Pd1 pecf594, by BD (1 mentions)
Cd4 pb, by BD (1 mentions)
Il 10 apc, by BD (1 mentions)
6 protocols using rorγt pe
1
Comprehensive Immune Cell Profiling
2
Multiparametric Flow Cytometry Analysis
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Flow cytometry Abs were purchased from BD Pharmingen, e-Bioscience and Biolegend (San Diego, CA). Single-cell suspensions, after anti-CD16/CD32 blocking, were stained for 25 min at 4°C with the following primary Abs or isotype controls: IgA-FITC (Clone C10-3), B220-PE (Clone RA3-6B2), CD138-APC (clone 281-2), CD5-PE (clone 53-7.3), CXCR5- PE-Cy7 (clone L138D7), PD1-FITC (clone J43), CD11b−APC (Clone M1/70), CD86-PE (Clone GL1), CD80-APC (Clone 16-10A1), CD11c-FITC (Clone HL3,), MHC-II-FITC (Clone 39-10-8), CD64-PerCP-Cy5.5 (clone X54-5/7.1), CD103-PE (Clone M290), CD19-PerCP (Clone 6D5), CD45-APC-Cy7 (Clone 30-F11), CD3-FITC (Clone 17A2), CD4-PE-Cy7 (Clone 4-5), TCRβ-PE (Clone H57-597), TCRδ-APC (Clone GL3). For intracellular IL-17A and FoxP3 staining, cells were fixed with Fix/Perm reagents (Cyto Fix/Perm, BD) for 30 min at 4°C and stained with IL-17A-PerCP-Cy5.5 (Clone TC11-18H10), RORγt-PE (Clone B2D) or FoxP3-PE (Clone MF23). Events were acquired on FACSCanto II (BD Biosciences) and were analyzed with Flowjo software version 7.6.1.
3
Flow Cytometry Immunophenotyping Protocol
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For flow cytometry analyses cells were stained with antibodies to detect surface expressed CD4 (CD4 Alexa 488; CD4 APC; CD4 Percp [BD Biosciences]) and intracellular expressed RORγT, IL‐10, IL‐17A, and IFN‐γ (RORγt PE, IFN‐γ FITC, IL‐17A Percp, IL‐10 PE‐ BD biosciences). Staining was performed with Fix/Perm and Perm/Wash from BD Biosciences according to the manufacturer's protocols. To monitor cytokine expression by flow cytometry, cells were incubated with the corresponding stimuli and stimulated for 4 h in the presence of PMA (50 ng/mL), Ionomycin (500 ng/mL) and 1.5 μL/1 mio cells Golgi‐StopTM (BD Biosciences) before staining. Cells were analyzed using FACS Calibur from BD Bioscience and data analysis was performed with FlowJo software from Treestar (San Carlos).
4
Multiparametric Immune Cell Profiling
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The following anti-human antibodies were used for the lineage the cocktail: CD3-FITC, CD4-FITC, CD8a-FITC, CD14-FITC, CD15-FITC, CD16-FITC, CD19-FITC, CD20-FITC, CD33-FITC, CD34-FITC, FcεRI-FITC, and CD203c-FITC (all from Biolegend). In addition, these anti-human antibodies were used: CD335 (NKp46)-PE/Cy7, CD294 (CRTH2)-PE, CD127-BV421 from Biolegend, CD56-APCeF780 from eBioscience, and CD117 (ckit)-APC from BD Bioscience. For intracellular transcription factor staining the following anti-human antibodies were used: Tbet-PE/CF594, GATA3-PE/Cy7, and RORγt-PE from BD Bioscience. Corresponding isotype control antibodies were used as controls.
5
Isolation and Analysis of Intestinal Immune Cells
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Spleens were collected and mashed in 70 μm cell strainers with complete culture media. Red blood cells were lysed with RBC lysis buffer (eBioscience). To isolate LP cells, the small intestine was cut into pieces and digested by a buffer with Dispase, DNase, and Collagenase. Intestinal epithelial cells were removed, and Percoll was used for the isolation of lymphocytes. For surface staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with a BD FACSAria II flow cytometer (BD Biosciences). For intracellular staining, a Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include APC anti-CD3, PE-Cy7 anti-CD4, FITC anti-CD8, PerCP-Cy5.5 anti-CXCR5, APC-Cy7 anti-PD-1, PE anti-B220, FITC anti-CD38, APC anti-GL7, FITC anti-IgA, PerCP-Cy5.5 anti-CD45, “Lin”: biotin anti-CD3 and biotin anti-CD19, a Zombie Aqua Fixable Viability Kit (Biolegend), RORγt-PE (BD Biosciences), and APC anti-biotin (Miltenyi Biotec). Flow cytometry data were analyzed with FlowJo.
6
Multiparametric Flow Cytometry of Immune Cells
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The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD304 (Nrp1)-PerCP eF710, c-Maf-EF660, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: CD25-BB515, CD44-PECy7, CD4-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, STAT3 (pY705)-A647, streptavidin-PECy7.
Live/dead fixable near-IR stain (ThermoFisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer's protocol (FOXP3 staining buffer set from eBioscience). For phosphorylation staining, cells were fixed with formaldehyde and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) or CytoFLEX (Beckman Coulter) and analyzed using FlowJo software (Tree Star).
Live/dead fixable near-IR stain (ThermoFisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer's protocol (FOXP3 staining buffer set from eBioscience). For phosphorylation staining, cells were fixed with formaldehyde and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) or CytoFLEX (Beckman Coulter) and analyzed using FlowJo software (Tree Star).
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