Anti ki67 mab sola15

Two different proliferation assays were performed; cells were washed prior to addition of IL2Rγ-chain cytokines in both assays. In the standard proliferation assay (figures 1c, 1d, supplementary figures 3c, 3d, 4a, 4b, 4c), cells were exposed to 5-bromo-2′-deoxyuridine (BrdU; 10 mM, BD) for 2 hours beginning 2 days after addition of IL2Rγ-chain cytokines, then fixed and permeabilized according to the manufacturer’s protocol for Cytofix/Cytoperm (BD), and stained with anti-Ki67 mAb (SolA15, eBioscience) and anti-BrdU mAb (Bu20a, Biolegend) as previously described [25 (link)]. For the simultaneous proliferation/cytokine production assay (figures 1e, 1f), cells were exposed to BrdU for 8 hours beginning 1 day after addition of IL2Rγ-chain cytokines. Cells were then washed and soluble anti-CD3 mAb (10ug/ml) or vehicle was added, along with the protein transport inhibitor Golgistop (BD), for 5 hours to stimulate cytokine production and intracellular accumulation. Cells were then fixed and stained for CD4, IL-17, Ki67, and BrdU.

Flow cytometry analysis was performed as previously described (17 (link)). The antibodies used in this study include CD8 (53–6.7), CD25 (PC61), CD45.1 (A20), IFNγ (XMG1.2), STAT5 pY694 (47/Stat5(pY694)), Thy1.1 (A20), and TNFα (TN3-19.12).These were purchased from BD Bioscience, Biolegend (San Diego, CA), and eBioscience (San Diego, CA). For analysis of phosphorylation of STAT5, we followed the manufacturer’s protocol using Lyse/Fix and PermIII buffer (BD Bioscience). To examine cellular proliferation, cells were fixed and permeabilized according to the manufacturer’s protocol for Cytofix/Cytoperm (BD Bioscience) and stained with anti-Ki67 mAb (SolA15, eBioscience). Alternatively, BrdU (10μm) was added one hour prior to harvest, and cells were analyzed for BrdU incorporation as previously described (17 (link)). For Foxp3 staining, we followed the protocol outlined in the Foxp3 kit (eBioscience). Flow cytometry was performed on BD LSRII and BD FACSAccuri. Data were analyzed using FlowJo software (TreeStar). In all experiments, initial gating of live cells was performed using forward scatter and side scatter parameters, and cells were then gated on live lymphocytes. Isotype and fluorescence minus one (FMO) controls were performed as required. For experiments assessing IL-2, we always included control conditions without IL-2 pulsing.