Computational protocol: Retrovirus-Specificity of Regulatory T Cells Is Neither Present nor Required in Preventing Retrovirus-Induced Bone Marrow Immune Pathology

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Protocol publication

[…] Cells were stained with directly conjugated antibodies to surface markers, obtained from eBiosciences (San Diego, CA, USA), CALTAG/Invitrogen (Carlsbad, CA, USA), or BD Biosciences. Four- and eight-color cytometry were performed on FACSCalibur (BD Biosciences) and CyAn (Dako) flow cytometers, respectively, and analyzed with FlowJo v8.7 (Tree Star, Ashland, OR, USA) or Summit v4.3 (Dako) analysis software, respectively. For detection of cytokine synthesis, cells were stained for surface markers and stimulated for 4 hr with PdBu and ionomycin (both at 500 ng/ml), in the presence of Monensin (1 μg/ml). Cells were then fixed and permeabilized with buffers from eBiosciences, before intracellular staining with IL-17A and IFN-γ-specific antibodies (eBiosciences). FoxP3 was detected by intranuclear staining with a FoxP3-staining kit (eBiosciences), according to the manufacturer's instructions. Ab-env122-141 tetramers (kindly provided by D. Gray, University of Edinburgh, UK) were prepared and used as previously described (). In brief, cells were incubated with PE-labeled Ab-env122-141 tetramers at 37°C in IMDM containing 5% FCS; this was followed by staining with antibodies to surface markers, before analysis. [...] Statistics were generated by Student's t test performed with SigmaPlot v10 software (Systat Software, San Jose, CA, USA). […]

Pipeline specifications

Software tools FlowJo, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Addison Disease, Anemia, Bone Marrow Diseases, Infection, Virus Diseases
Chemicals Receptor-CD3 Complex, Antigen, T-Cell