ccl2 antibody staining cells  (Revvity)

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Shared pathways significantly upregulated in endothelial cells treated with EVs.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Protein-Protein interactions, Migration, Chemotaxis Assay, Variant Assay, Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Endothelial cells were left untreated or treated for 16 hrs with LPS (1μg/mL) or EVs derived from non-infected or Mtb -infected macrophages. (A) Cells were stained with FITC conjugated anti-mouse TLR2 antibody or FITC conjugated anti-mouse IgG1 antibody as an isotype control. (B) Cells were surface-stained with FITC-labeled rat anti-mouse VCAM1 or FITC labeled anti-rat IgG2a antibody as an isotype control. (C) Cells were permeabilized and stained for intracellular CCL2 using PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gates were set to approximately 1% for isotype control and were maintained for all subsequent analysis. RC: untreated cells, RvEV: Treatment with EVs from H37Rv-infected macrophages, UnEV: Treated with EVs from non-infected macrophages. Data is representative of the protein expression from three independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Staining, Control, Labeling, Expressing

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: (A) SVEC4-10 cell monolayers were left untreated or stimulated for 3 hrs with EVs derived from non-infected or Mtb -infected mice. CFSE-labeled mouse BMMs were added to SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged. The number of BMMs in seven randomly selected fields were counted and the total number of cells for each condition defined. The data is the average of three independent mouse Mtb infections +SD with (*) indicating a p value < 0.05 compared to RC. (B) Quantitative RT-PCR was performed on endothelial cells that were left untreated or treated for 4 hours with EVs derived from uninfected or Mtb -infected macrophages. Total RNA was extracted followed by cDNA synthesis. Fold change of gene expression was calculated by comparative Ct method. Data is from two independent mouse Mtb infections. (C) Scatter plots of flow cytometry analysis of CCL2 expression. Endothelial cells were left untreated or treated for 16 hours with EVs derived from non-infected or Mtb -infected macrophages. Permeabilized cells were stained with PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gate was set for isotype control and was maintained for all subsequent analysis. RC: untreated cells. Un-EV: serum-derived EVs from uninfected mice, D7-EV, D14-EV, D21-EV: serum derived EVs from mice infected for 7, 14 and 21 days respectively. Data is representative of the CCL2 expression from two independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Labeling, Quantitative RT-PCR, cDNA Synthesis, Gene Expression, Flow Cytometry, Expressing, Staining, Control