F TEMs (best gate, red) and TIE2?monocytes (bottom gate, black). Post-sort purity check (suitable dot plots) show higher purities, 94.five ?0.eight for TEMs (n ?5 samples). F. RT-PCR traces showing that expression of TIE2 is present in TEM samples after 25 cycles but is absent in TIE2?monocytes. n ?eight CLI individuals, TIE2?and TIE2?samples analysed in triplicate. G. (i) Gating on the entire monocyte population (red gate) for phenotyping in line with CD14 and CD16 expression shows the common distribution of classical (CD14��CD16?bottom correct quandrant), intermediate (CD14��CD16? best ideal quadrant) and non-classical (CD14�CD16? best left quadrant) monocytes. (ii) Gating of TEMs (red gate) for phenotyping according to CD14 and CD16 expression shows that the majority of these cells express CD16 and are, for that reason, identified within either the intermediate or non-classical subset.TEMs have proangiogenic activity and respond to angiopoietin stimulation TEMs are identified to have proangiogenic functions both in vitro and in vivo (Coffelt et al, 2010; De Palma et al, 2005) however the activity of TEMs isolated from aged CLI sufferers with several co-morbidities has not previously been investigated. TEMs isolated in the blood of CLI sufferers and co-cultured with HUVECs on Matrigel exhibited a higher capacity to enhanceHUVEC tubule formation compared with TIE2?monocytes in the similar folks ( p 0.05, Fig 3A and B). Possessing identified differences in the numbers and proangiogenic activity of circulating and muscle-resident TEMs amongst CLI and controls, we next measured a panel of circulating angiogenic and proinflammatory elements within the plasma of CLI patients and compared this with controls (Table two). The levels of angiopoietin-2 (ANG2, a TIE2 ligand), vascular endothelial growth aspect?2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.EMBO Mol Med (2013) five, 858?embomolmed.orgResearch iNOS Inhibitor Storage & Stability ArticleAshish S. Patel et al.Figure two. Quantification of TIE2R macrophages in human muscle specimens. A. Muscle specimens had been enzymatically digested and analysed by flow cytometry. Gating (red gates) of CD45 good cells (i) followed by exclusion of lineage (CD19, CD56, CD3) constructive cells (ii), exclusion of doublets (iii) and collection of CD68?macrophages (iv). B. Gate for TIE2 expression set based on staining with FMO sample (left). Instance TIE2 staining of cells from healthy muscle (middle) and ischemic muscle (correct) showing a greater proportion of TIE2?macrophages within the ischemic compared with typical tissue. C. Histogram (gated on CD68?macrophages) showing higher expression of TIE2 in macrophages from ischemic (red) compared with wholesome (blue) muscle. D. Flow cytometry evaluation of digested muscle specimens shows greater proportion of CD68?macrophages expressing TIE2 in distal ischemic muscle compared with proximal healthier muscle biopsies from CLI patients (11.three ?2.2 vs. 4.5 ?1.three , respectively). 0.05 by paired t-test. E. H E sections of normoxic (major) muscle compared with ischemic (bottom) muscle which shows loss of your standard muscle architecture and cellular infiltrate. Scale bars represent 50 mm. F. Immunofluorescence stains of a section of ischemic muscle displaying nucleated cells (blue) expressing CD14 (green) and TIE2 (red) close to a blood vessel lined with TIE2-expressing endothelial cells (arrows). Merged image shows TEMs (L-type calcium channel Agonist Storage & Stability orange, arrows). G. Section of ischemic muscle displaying nucleated cells (blue) expressing CD68 (green) and TIE2 (red). Merged imag.