Computational protocol: iNKT cell development is orchestrated by different branches of TGF-β signaling

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Protocol publication

[…] CD24-FITC (clone M1/69) mAbs, CD122-PE (clone TM-β1), Ki67-PE (clone B56), NK-1.1-PerCP-Cy5.5 (clone PK136), Ly9.1, TCR-β-FITC (H57-597) annexin V-APC and 7-AAD, NKG2A/C/E (20D5), NKG2D (C7), and Ly49C/I (5E6) were obtained from BD. CD127-PE (clone A7R34), NK-1.1-PE-Cy7 (clone PK136), T-bet-Alexa Fluor 647 (clone eBio4B10), CD44-Alexa Fluor 700 (clone IM7), IL-4 (11B11), and IFN-γ (XMG1.2) were purchased from eBioscience. CD1d-α-GalCer tetramers were produced with streptavidin-APC (BD) or streptavidin-PE-Cy7 (eBioscience) and used for staining as previously described (). Anti–TGF-βRII-biot was purchased from R&D Systems. Anti-Smad4 (EP618Y) was purchased from Abcam and monoclonal and anti-Tif-1γ (5Tγ3E9) were gifts from Institute of Genetics and Molecular and Cellular Biology (Strasbourg, France). For intracellular staining, cells were activated with 500 ng/ml PMA and 5 ng/ml ionomycin (Sigma-Aldrich), and then resuspended in Cytofix/Cytoperm solution from a Fixation/permeabilization kit (BD) and stained according to the manufacturer's instructions. Flow cytometry and cell sorting was performed using a two-laser FACSAria (BD) cell sorter/analyzer and analyzed using either BD FACSDiva software v5.0.1 (BD) or FlowJo (Tree Star, Inc.). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry