2011;117:6083C6090. progression of certain cancers, including haematopoietic malignancies3C6. Ribosomal defects commonly impair HSC and erythroid progenitor function7C11. However, it is not clear whether these defects reflect a catastrophic reduction in protein synthesis below the level required for cellular homeostasis or whether HSCs require highly regulated protein synthesis. Methods for measuring protein synthesis have depended upon the incorporation of radiolabeled amino acids, amino acid analogues12, or puromycin13C15 into nascent polypeptides in cultured cells. However, somatic stem cells profoundly change their properties in culture16 necessitating the analysis of protein synthesis in rare cells in vivo. A new fluorogenic assay using O-propargyl-puromycin (OP-Puro) has been developed to image protein synthesis in vivo17. OP-Puro, like puromycin, is taken up by cells in vivo, entering ribosome acceptor sites and incorporating into nascent polypeptides17. An azide-alkyne reaction can be used to fluorescently label OP-Puro to quantitate protein synthesis in individual cells17. We adapted this approach to quantify protein synthesis by haematopoietic cells using flow cytometry. HSCs synthesize less protein per hour We administered a single intraperitoneal injection of OP-Puro (50mg/kg body mass) then sacrificed mice one hour later and isolated bone marrow cells. We did not detect toxicity, signs of illness, changes in bone marrow cellularity, or changes in the frequencies of CD150+CD48?Lineage?Sca-1+c-kit+ (CD150+CD48?LSK) HSCs18, Annexin V+ bone marrow cells, Annexin V+ HSCs, or dividing HSCs (Extended Data Fig. 1aCe). Bone marrow cells from OP-Puro treated mice exhibited a clear increase in fluorescence relative to untreated mice (Fig. 1a). The translation inhibitor, cycloheximide, profoundly blocked OP-Puro incorporation by bone marrow cells in culture (Fig. 1b). Incorporation of the methionine analogues, L-homopropargylglycine (HPG) and L-azidohomoalanine (AHA), into bone marrow cells, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and Gr-1+ myeloid cells correlated with OP-Puro incorporation in culture (Fig. 1cCf). Open in a separate window Figure 1 Quantifying protein synthesis in haematopoietic cells in vivoa, OP-Puro incorporation in bone marrow cells in vivo one hour after administration. b-d, Chlorpropamide OP-Puro (b), HPG (c), and AHA (d) incorporation in bone marrow cells in culture was inhibited by cycloheximide (CHX). d, Bone marrow cells from mice treated with OP-Puro in vivo exhibited normal LAMC1 antibody AHA incorporation in culture, indicating OP-Puro did not block protein synthesis. e,f OP-Puro versus HPG (e; n=4 mice from 2 experiments) or AHA (f; n=3 mice from 3 experiments) incorporation by haematopoietic cells in culture. g, OP-Puro incorporation in CD150+CD48?LSK HSCs and unfractionated bone marrow cells one hour after administration in vivo. h, Protein synthesis in various haematopoietic stem and progenitor cell populations relative to Chlorpropamide unfractionated bone marrow cells (n=15 mice from 9 experiments). Extended Data Fig. 1j shows the data from Fig. 1h using a log2 scale. Data represent means.d. Statistical significance was assessed using two-tailed Students t-tests (e-f) and differences relative to HSCs (h) were Chlorpropamide assessed using a repeated measures one way ANOVA followed by Dunnetts test for multiple comparisons (*, p<0.05; **, p<0.01; ***, p<0.001 relative to bone marrow; ###, p<0.001 relative to HSCs). HSCs incorporated less OP-Puro than most other bone marrow cells from the same mice (Fig. 1g). This suggested that HSCs synthesize less protein per hour than most other haematopoietic progenitors. CD150?CD48?LSK multipotent progenitors (MPPs)19 exhibited similar OP-Puro incorporation as HSCs (Fig. 1h); however, the mean rate of OP-Puro incorporation was significantly higher in unfractionated bone marrow cells, CMPs, GMPs, megakaryocyte-erythroid progenitors (MEPs), Gr-1+ myeloid cells, B220+IgM?CD43+ pro-B cells, B220+IgM?CD43? pre-B cells, B220+IgM+ B Chlorpropamide cells, CD3+ T cells, and CD71+Ter119+ erythroid progenitors (Fig. 1h). Extended Data Figures 1fCi show markers, gating strategies, and OP-Puro incorporation histograms for each cell population. To test whether reduced OP-Puro incorporation into HSCs reflects OP-Puro efflux by the Abcg2/Bcrp1 transporter we administered Chlorpropamide OP-Puro to HSCs continued to exhibit significantly lower mean rates of OP-Puro incorporation as compared to most other progenitors (Fig. 2a), similar to the lowest levels observed among bone marrow cells (Fig. 2b). Open in a separate window Figure 2 Lower rate of OP-Puro incorporation by HSCs does not reflect efflux or proteasomal degradationa,b, OP-Puro fluorescence in haematopoietic cells from mice have a hypomorphic mutation in the Rpl24 ribosome subunit, reducing protein synthesis in multiple cell types by 30% in culture3,4,26. Adult mice are grossly normal but are 20% smaller than wild-type mice and have mild pigmentation and skeletal abnormalities26. These.