Cfse proliferation dye

Mouse and human cytokines used for in vitro polarizations as well as anti-human CD3 and anti-human CD28 were purchased from Biolegend. Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell. Anti-CK2α and anti-CK2β antibodies for detection by flow cytometry were purchased from AbCam and Calbiochem, respectively. Secondary antibodies for flow cytometry were purchased from Jackson ImmunoResearch. Flow cytometry antibodies against mouse CD4, IL-17A, IFN-γ, CD25 and CD45.1 and human CD3, CD4 and Foxp3 were purchased from Biolegend. Flow cytometry antibodies against mouse CD44, Foxp3 and GM-CSF and human IL-17A were purchased from eBioscience. Aqua Live/Dead Viability dye, CFSE proliferation dye and 2-NBDG were purchased from ThermoFisher Scientific. Flow cytometry antibodies and isotype controls for phosphorylated S6, Akt, STAT3, STAT5 and SMAD2/3 were purchased from Cell Signaling. Immunoblotting antibodies against phosphorylated T389, S371 and total S6 Kinase p70 and phosphorylated S129, S473 and total Akt were purchased from Cell Signaling, and antibody against mouse β-actin was purchased from abcam.

Murine cytokines used for in vitro polarizations were purchased from Biolegend. Anti-mouse CD3, anti-mouse CD28 and neutralizing antibodies to murine IL-4 and IFN-γ were purchased from BioXCell. Antibodies to CK2α, phosphorylated S129 Akt, total Akt, phosphorylated T24 FoxO1 and total FoxO1 were purchased from Cell Signaling. Antibody to mouse β-actin was purchased from Abcam. Flow cytometry antibodies against mouse CD3, CD4, CD8, CD25, IL-17A and IFN-γ were purchased from Biolegend. Flow cytometry antibodies against mouse CD44, CD62L, CD69, Foxp3 and GM-CSF were purchased from eBioscience. Flow cytometry antibodies for phosphorylated STAT3, STAT5, S6 and Akt were purchased from Cell Signaling. Aqua Live/Dead Viability dye and CFSE proliferation dye were purchased from ThermoFisher Scientific.

Spleens were dissociated in FACS buffer (0.5% BSA in PBS) to generate single-cell suspensions. Splenocytes were passed through a 70um filter and then RBC lysed using ACK lysis buffer (0.15M NH4CL, 1mM KHC3, 0.1mM Na2EDTA) for 5 minutes at room temperature. Samples were stained with Ghost Dye Violet 510 (Tonbo) for 20 minutes at 4°C, then stained with the following antibodies: CD4 (GK1.5), B220 (RA3–6B2), CD8b (YTS156.7.7), CXCR3 (CXCR3–173), KLRG1 (2F1/KLRG1), Ki67 (SolA15), CD44 (1M7), CD3 (145–2C11), CD11c (N418), MHC-II (M5/114.15.2), CD11b (M1/70), XCR1 (ZET), DCIR2 (33D1), CD25 (PC61), CD45.1 (A20), CD45.2 (104), NK1.1 (PK136), Ly6G (1A8). In some experiments, cells were labeled with CFSE proliferation dye (ThermoFisher) following the manufacturer’s protocol. For intracellular staining, samples were fixed and permeabilized using the eBioscience FoxP3 staining kit (ThermoFisher). Samples were processed using an LSR Fortessa Analyzer (BD) and analyzed using FlowJo software.