To detect protein synthesis rate in vivo, primary or stressed mice were injected intraperitoneally with OP-Puro (Cat# HY-15680, MCE; 50mg/kg body weight, pH6.4–6.6 in PBS) for 1 hour before euthanasia (Signer et al., 2014 (link)). Total BM cells were harvested and live stained with cell surface markers for HSCs/MPPs after a quick RBC lysis. Cells were then fixed with BD Cytofix solution for 20min on ice. After washing with BD Perm/Wash buffer, cells were permeabilized with BD Cytoperm Plus solution for 10min on ice, followed by refixing in Cytofix solution for 5min. The azide-alkyne reaction was performed using Click-iT plus OPP Alexa Fluor 647 or 488 kit (Cat# C10458, Invitrogen) for 30min at room temperature. Cells were then washed and resuspended in flow buffer, and analyzed by on a BD FACS Fortessa flow cytometer.
For ex vivo analysis, OP-Puro was added to cell culture at the final concentration of 20uM for 1 hour at a 37°C incubator. The azide-alkyne reaction was performed as described above.
For ex vivo analysis, OP-Puro was added to cell culture at the final concentration of 20uM for 1 hour at a 37°C incubator. The azide-alkyne reaction was performed as described above.