Op puro

For measuring protein synthesis in vitro, Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (Thermo Fisher Scientific) was used as indicated. T cells were incubated with OP-puro in culture for 30 min before or at various time points after anti-CD3/CD28 stimulation, and then cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences). The azide-alkyne cycloaddition was performed according to the manufacturer’s instructions. After the 30-min reaction, the cells were washed and analyzed by flow cytometry.
For in vivo analysis, OP-puro (MedchemSource; 50 mg per kg of body mass; pH 6.4–6.6 in PBS) was injected intraperitoneally into mice that were immunized with 100 μg of freshly prepared EndoFit ovalbumin (OVA, InvivoGen) intramuscularly 48 hours earlier. One hour later mice were euthanized, unless indicated otherwise. Splenocytes were collected and 2×10⁶ cells were stained with combinations of antibodies against cell-surface markers as described in the figures. After washing, the cells were fixed, permeabilized, and the azide-alkyne cycloaddition was performed according to the manufacturer’s instructions. Mean OP-puro fluorescence reflected absolute fluorescence values for each cell population from multiple independent experiments.