Journal: Scientific Reports

Article Title: Precision Targeting of Tumor Macrophages with a CD206 Binding Peptide

doi: 10.1038/s41598-017-14709-x

Figure Lengend Snippet: UNO specificity for CD206. ( a ) Change in fluorescence anisotropy of FAM-UNO (dotted line) and FAM-UNO in DTT (solid line) while incubating with mouse recombinant CD206. ( b ) Change in fluorescence anisotropy of FAM-CSPGAK with mouse recombinant CD206 (solid line) or with CD163 (dotted line) and of FAM-CPMTDNE (control) with CD206 (dashed line). ( c ) FAM-UNO binds selectively to CCR2 + macrophages collected from the peritoneal cavity of 4T1 tumor-bearing mice, as 94.3% of FAM + cells are CCR2 + cells. The analysis was done gating for the FAM + population (left panel). In these 4T1 tumor bearing mice, 58% of peritoneal cells are macrophages, i.e CCR2 + cells (right panel). ( d ) FAM-UNO binding to peritoneal cells is inhibited by preincubating with 10 µg/mL of anti-CD206, whereas the preincubation with anti CD206 antibody had no effect on FAM-LyP-1 binding. In panels a and d are shown representative graphs from three independent experiments. In panels c and d are shown results from three independent experiments (n = 3 mice) and bars of panel d represents mean + SEM.

Article Snippet: The recombinant mouse macrophage mannose receptor rmMMR/CD206 (R&D Systems, catalog number: 2535-MM-050) and recombinant mouse CD163 (R&D Systems, catalog number: 7435-CD-050) were reconstituted at 1 mg/ml in IB, aliquoted and stored at −20 °C.

Techniques: Fluorescence, Recombinant, Control, Binding Assay

Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

doi: 10.1681/ASN.2013060675

Figure Lengend Snippet: Evaluation of M2-like Mϕ-related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data represent mean±SEM values. Day 6 (CSF-1) indicates that every mouse in each genotype group was administered 5.0 μg CSF-1. n=6 for each group. *P<0.05 compared between the +/+, +/op, and op/op groups at the same time points; †P<0.05 compared with day 0 in each group; §P<0.05 compared with day 6 in each group. (B) Fluorescence immunohistochemical distribution of CD163 and CD206 expression in mouse kidneys harvested on days 0 and 6. CD163 and CD206 double positive Mϕs are shown in the interstitial cells of the corticomedullary junction in the +/+ and +/op groups; however, neither protein was expressed in the op/op group on day 0 or 6. The expression of CD163 and CD206 was increased by CSF-1 treatment. Blue, nucleus; green, CD206; red, CD163. Original magnification, ×400; ×800 in inset.

Article Snippet: For the detection of M φ -related genes, we used probe sets for Emr1 (Mm00802529_m1), Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il 6 (Mm00446190_m1), Cd 163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il 10 (Mm00439614_m1).

Techniques: Expressing, Quantitative RT-PCR, Control, Fluorescence, Immunohistochemical staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

doi: 10.1681/ASN.2013060675

Figure Lengend Snippet: Flow cytometric analysis of renal Mϕs. (A) Gating strategy for CD11b+F4/80+ of renal mononuclear cells. The population of F4/80hiCD11b+ cells was determined from the representative graph. Isotype controls of both F4/80 and CD11b proteins are shown as red dot plots (upper panel). Back gates for live cells and CD45 are also shown. Most F4/80hiCD11b+ cells were CD45-positive and 7AAD-negative (lower left panel). The dotted line shows the isotype control of CD45 protein (lower right panel). (B) The CD11cintLy6Chi (upper panel) and CD163+CD206hi (lower panel) subpopulations of CD11b+F4/80+ cells are shown. Isotype controls for CD11c and Ly6C (upper panel) and CD163 and CD206 (lower panel) are shown on each graph as red dotted lines. (C) Quantification of CD11b+F4/80+CD11cintLy6Chi (upper left panel) and CD11b+F4/80+CD163+CD206hi Mϕs (lower left panel). There were no significant differences between +/+ and op/op mice except on day 0, and treatment with CSF-1 did not change the characteristics of those populations; op/op mice had a significantly lower number of CD11b+F4/80+CD163+CD206hi Mϕs than +/+ mice on days 0 and 6. The number of CD11b+F4/80+CD163+CD206hi Mϕs was increased after CSF-1 treatment in op/op mice. The median fluorescent intensities (MFIs) of Ly6C, CD11c, CD206, and CD163 in CD11b+F4/80+ cells are shown in right panel; op/op had lower intensities of CD206 and CD163 staining than +/+ mice, but CSF-1 treatment increased CD206 intensity. The data represent mean±SEM values. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 μg CSF-1. n=4 for each group. *P<0.05 compared with the +/+ group; †P<0.05 compared with day 0 in each group; §P<0.05 compared with day 6 in each group. (D) M1-like Mϕ expression. Representative FACS histograms of either CD11c or Ly6C in CD11b+F4/80+ cells are shown. Dotted lines show isotype controls of CD11c and Ly6C proteins. (E) M2-like Mϕ expression. Representative FACS histograms of either CD206 or CD163 in CD11b+F4/80+ cells are shown. Dotted lines show isotype controls of CD206 and CD163 proteins.

Article Snippet: For the detection of M φ -related genes, we used probe sets for Emr1 (Mm00802529_m1), Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il 6 (Mm00446190_m1), Cd 163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il 10 (Mm00439614_m1).

Techniques: Control, Staining, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

doi: 10.1681/ASN.2013060675

Figure Lengend Snippet: Evaluation of the effect of M2Mϕ transfusion on crystal formation. In each graph, the data from day 6 experiments of each genotype (described previously) were used as the control to compare the differences between groups treated with M2Mϕ and those groups not treated. Day 6 (M2Mϕ) indicates that every mouse in each genotype group was transfused with 1.0×106 cells of M2Mϕs. Data represent means±SEMs. (A) Representative micrographs of renal sections harvested on day 6 from M2Mϕ-transfused mice. Each genotype had the same intratubular CaOx crystals from the corticomedullary junction to papilla. n=5 for each group. Original magnification, ×40; ×400 in inset. (B) Quantitative estimation of renal and urinary CaOx crystals. (Left panel) The ratio of the area containing renal crystal deposition. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using Image Pro Plus. (Right panel) The number of urinary crystals per high-powered field; 24-hour urine samples were collected in a metabolic cage, and CaOx crystals were counted at ×400 magnification. n=5 for each group. (C) Evaluation of crystal binding and related gene expressions. The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means±SEMs. n=5 for each group. (D) Flow cytometric analysis of renal Mϕs. The numbers of CD11b+F4/80+CD11cintLy6Chi (left panel) and CD11b+F4/80+CD163+CD206hi (right panel) Mϕs were calculated by flow cytometry. The gating strategy was the same as the strategy used in Figure 7 (described above). n=4 for each group. *P<0.05 compared with the +/+ group.

Article Snippet: For the detection of M φ -related genes, we used probe sets for Emr1 (Mm00802529_m1), Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il 6 (Mm00446190_m1), Cd 163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il 10 (Mm00439614_m1).

Techniques: Control, Crystallization Assay, Binding Assay, Expressing, Quantitative RT-PCR, Flow Cytometry