Single cell suspensions were stained with antibodies for CD4 (GK1.5), γδ TCR (GL3), IL-17A (TC11-18H10.1), IFN-γ (XMG1.2) (Biolegend) and TGFβRII (R&D Systems). For intracellular staining, cells were restimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for 4h and Brefeldin A (10 µg/ml) was added for the last 3h. Cells were stained with fixable Live/Dead stain (Invitrogen) followed by staining of surface markers, fixed with formaldehyde, permeabilized and stained for intracellular cytokines. Acquisition was performed on a LSR Fortessa (BD Biosciences) and data analysis was performed with Flowjo software (Tree Star).
Tgfβrii
Manufactured by R&D Systems
Sourced in Canada
TGFβRII is a recombinant human Transforming Growth Factor Beta Receptor II protein. It is a cell surface receptor that binds to transforming growth factor-beta (TGF-beta) and plays a role in TGF-beta signaling.
Automatically generated - may contain errors
Lab products found in correlation
Fixable live dead stain, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Ifn γ xmg1.2, by BioLegend (2 mentions)
Rabbit serum, by Merck Group (1 mentions)
Fortessa 2, by BD (1 mentions)
Fix perm kit, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Aria 3, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Goat anti mouse igg, by BioLegend (1 mentions)
Nkp30, by BioLegend (1 mentions)
Dnam 1, by BioLegend (1 mentions)
4 protocols using tgfβrii
1
Single-cell Cytokine Profiling Workflow
2
Intracellular Cytokine Staining Protocol
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For intracellular staining of cytokines, cells were stimulated with PMA (Sigma-Aldrich)/ionomycin (Emdchemicals) in the presence of Brefeldin A (Sigma-Aldrich) for 3.5 hr at 37°C. Cells were washed and incubated for 20 min with rabbit serum (Sigma-Aldrich) prior to staining for extracellular antigens in 5% FCS/1% BSA in PBS for 30 min at 4°C. All antibodies were purchased from eBioscience with the exception of ICOS (Biolegend), TGF-βRII (R&D Systems), and T1/ST2 (MD Bioproducts). Cells were washed, fixed, and permeabilized using Fix/Perm kit (eBioscience) before being stained for intracellular antigens. Analysis was performed with Fortessa II and cell sorting on Aria III (BD Biosciences).
3
Single-cell Cytokine Profiling Workflow
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Single cell suspensions were stained with antibodies for CD4 (GK1.5), γδ TCR (GL3), IL-17A (TC11-18H10.1), IFN-γ (XMG1.2) (Biolegend) and TGFβRII (R&D Systems). For intracellular staining, cells were restimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for 4h and Brefeldin A (10 µg/ml) was added for the last 3h. Cells were stained with fixable Live/Dead stain (Invitrogen) followed by staining of surface markers, fixed with formaldehyde, permeabilized and stained for intracellular cytokines. Acquisition was performed on a LSR Fortessa (BD Biosciences) and data analysis was performed with Flowjo software (Tree Star).
4
Phenotypic Characterization of TGF-β DNR Cells
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Cell phenotype, transduction efficiency, activation, and exhaustion of TGF-β DNR transduced cells and their nontransduced counterparts were determined by flow cytometry, using the following cell surface markers: CD3, CD56 (BioLegend, San Diego, CA), TGF-β RII (“wildtype” R&D, Minneapolis, MN), TGF-β RII (“DNR” Cambridge, UK), goat-anti mouse IgG, CD16, NKG2D, DNAM-1, NKp30, NKp46, CCR2, and CX3CR1 (BioLegend, San Diego, CA and BD Biosciencees, Franklin Lakes, NJ). Where reported, MFI was calculated from the geometric mean.
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