OP-Puro Labeling of Newly Synthesized Proteins

O-Propargyl Puromycin (NU-931-5; Jena Bioscience) was dissolved in DMSO, further diluted in PBS (10 mg/ml) and injected IP (50 mg/kg mouse weight). 1 hour after injection mice were euthanized by cervical dislocation and the organs of interest were collected. 7 × 10⁶ cells were stained with mixtures of antibodies directed against cell surface markers. Each staining lasted approximately 30′ and was performed on ice protected from direct light. Next, cells were fixed and permeabilized using the FoxP3 Fixation/Permeabilization kit (00-5521-00; eBioscience). For OP-Puro labeling, Azide-AF647 was chemically linked to OP-Puro through a copper-catalyzed azide-alkyne cycloaddition. In short, 2.5 μM azide-AF647 (A10277; Invitrogen) is dissolved in the Click-iT Cell Reaction Buffer (C10269; Invitrogen) containing 400 μM CuSO4. Immediately after preparation, cells are incubated with this mixture on room temperature. After 10′ incubation, the reaction is quenched by addition of PBS supplemented with 5% heat-inactivated fetal calf serum and 5 mM EDTA. Cells are washed twice to remove unbound azide-AF647 (Adapted from^{40 (link)}).