Protein Translation Assay Protocol

The protein translation assay was adapted from ref. ^{50 (link)}. First, single-cell suspensions were prepared as described above (‘Flow cytometry’). Then, the cells were washed and plated at 1 million cells per well in a V-bottom 96-well plate in methionine-free R10 (Gibco, A1451701) supplemented with 10% FBS, 1× non-essential amino acids (Gibco, 11140050), 10 mM HEPES (Gibco, 15630080, 7.2 to 7.5), 2 mM l-glutamine (Gibco, 25030081), 100 U ml⁻¹ penicillin–streptomycin (Gibco, 15140122) and 14.3 μM beta-mercaptoethanol. The cells were rested at 37 °C for 3 h, then 400 μM Click-iT HPG (Invitrogen, C10186) was added. After 3 h, cells were stained with viability dye and surface antibody cocktail as described above (‘Flow cytometry’). Then, the cells were fixed and permeabilized (BD, 51–2090KZ) for 20 min at room temperature (~22 °C), followed by one wash with perm wash (BD, 51-2091KZ) and one wash with PBS. Next, the Click-iT reaction was performed according to the manufacturer’s protocol (Invitrogen, C10641). Samples were analyzed as described above (‘Flow cytometry’).