pe anti a2b5  (Miltenyi Biotec)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing

doi: 10.1074/mcp.RA120.002102

Figure Lengend Snippet: Validation of OPC isolation method and overview of the ageing OPC proteome. A , Boxplot showing the log 2 protein intensity for PDGFRA in neonate, young and aged OPCs ( n = 6). B , Graph showing flow cytometric analysis of purity of freshly isolated OPCs at the three different ages shown as a percentage of live cells ( n = 4, Mean ± S.E.M. shown, statistics shown for A2B5 + MOG, Kruskal-Wallis p = 0.038*, Dunn's Multiple test comparison between neonate and young p = 0.0172, neonate and old p = 0.34 and young and old p = 0.60). C , Scatter plot showing the correlation of the log 2 changes of all protein groups among the 2 multiplexes. D , Graph showing the number of protein groups detected per age group. A total of 7481 protein groups were detected with 1% FDR. E , Venn diagram of the proteins identified with high confidence in multiplex. F , G , Graphs showing the relationship between protein level and transcript level for young ( F ) and aged ( G ) OPCs. Scatter plots show the representation of the correlation coefficients (Biweight midcorrelation coefficient) and the corresponding adjusted p values of significance ( p values were plotted as negative log 10 . Threshold (red dotted line) corresponds to FDR adjusted p value 0.05). Boxplots show the distribution of correlation coefficients for each detected protein.

Article Snippet: Then cells were pelleted and re-suspended in primary antibodies for 30 min except for A2B5-PE (Miltenyi, 130–093-581), which was added in the last 10 min of the incubation.

Techniques: Biomarker Discovery, Isolation, Comparison, Multiplex Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing

doi: 10.1074/mcp.RA120.002102

Figure Lengend Snippet: Validation of the ageing proteome. A , Heatmap showing the Z score expression of 4 randomly chosen proteins. Z score was calculated and heatmaps indicates the mean Z score for the six independent biological replicates of the proteomics dataset. Lower expression is represented in blue, and higher expression is shown in yellow. B , Western blotting images validating the proteome expression analysis in acutely isolated OPCs ( n = 3–6, Mean ± S.E.M. shown) C , Graphs show the densitometry quantification for PADI2, CRYAB, MOBP and FABP ( n = 3–4). D , E , Immunocytochemistry validation and quantification of the intensity of selected proteins per A2B5 + cell after 7 DIV. Scale bar 100 μm ( n = 3, Mean ± S.E.M. shown).

Article Snippet: Then cells were pelleted and re-suspended in primary antibodies for 30 min except for A2B5-PE (Miltenyi, 130–093-581), which was added in the last 10 min of the incubation.

Techniques: Biomarker Discovery, Expressing, Western Blot, Isolation, Immunocytochemistry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing

doi: 10.1074/mcp.RA120.002102

Figure Lengend Snippet: Changes in myelin proteins and cholesterol metabolism related proteins in aged OPCs. A , Heat map showing the increased expression of myelin proteins in aged OPCs. Z score indicates the mean of six independent biological replicates of the proteomics dataset. Lower expression is represented in blue, and higher expression is shown in yellow. B , Representative Western blotting showing myelin protein expression in A2B5 + OPCs and MOG + oligodendrocytes from different ages. C , Representative immunocytochemistry images of purified OPCs cultured for 6 days in vitro and immunostained for CLDN11 and MOG. D , Bar graphs indicating IHC quantification of myelin protein intensity per A2B5 + cells across the different age groups (each biological replicate is represented by a single dot) Scale bar 100 μm ( n = 3, Mean ± S.E.M. shown). E , F , Heat maps showing the expression changes for proteins involved in cholesterol biosynthesis or cholesterol transport, respectively. Z score indicates the mean of six independent biological replicates and lower expression is shown in blue, and higher expression is shown in yellow.

Article Snippet: Then cells were pelleted and re-suspended in primary antibodies for 30 min except for A2B5-PE (Miltenyi, 130–093-581), which was added in the last 10 min of the incubation.

Techniques: Expressing, Western Blot, Immunocytochemistry, Purification, Cell Culture, In Vitro