sema3a protein concentration  (Elabscience Biotechnology)

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 2. EV-transported Sema3A enhances vascular permeability through NRP1. (a) Sema3A concentrations were measured by ELISA in purified EVs from HEK293T, GSC#1, #4 and #9. (b) Flow-cytometry analysis of Sema3A surface expression in purified EVs from GSC#1, #4 and #9. PE-conjugated isotype control (IgG) is shown in grey. (c) Confocal analysis of purified GSC#1-shed EVs stained with Alix (green) and Sema3A (S3A, green). Nucleic acid staining is shown in blue (DAPI). Scale bar: 500 nm. (d) Electronic microscopy analysis of purified GSC#4-secreted EVs stained by immunogold for CD63 and Sema3A. Scale bars: 50 nm. (e) Western-blot analysis for Sema3A, CD9 and Rab4 in purified GSC#4-secreted EVs (EV#4) either untreated (−) or subjected to proteinase K treatment (Prot K, +). (f) EVs were purified from conditioned medium of GSC#4 that received either non-silencing RNA (siC, blue bar) or siRNA targeting Sema3A (siS3A, black bar). Sema3A concentrations were measured by ELISA. **Po0.01. (g, h) EVs as prepared in (f) were administered to measure endothelial permeability g) in vitro and (h) in vivo (n = 6). Results are expressed as fold change to untreated conditions ( −and PBS, respectively). Insert panel shows RT-PCR for Sema3A and Actin (ACTB) in GSC#4. *Po0.05; **Po0.01. (i) Permeability assays were performed in non-silencing (siC) or siRNA targeting NRP1 (siNRP1)-transfected endothelial cells (ECs). Two days after transfection, ECs were treated with purified GSC#4-secreted EVs (EV#4, green) or control vehicle (Ctrl, red). Insert panel shows RT-PCR for NRP1 and Actin (ACTB), to assess siNRP1 efficiency in ECs. **Po0.01. (j) ECs were pre-treated with blocking antibodies (25 mg/ml) preventing Sema3A (aNRP1A) or VEGF-A (aNRP1B) binding to NRP1 receptor. Human immunoglobulins (IgG) were used as a control with the same concentration as blocking antibodies. ECs were then exposed to purified GSC#4-shed EVs. **Po0.01. (k) Mice received intradermic injections of blocking (175 mg/ml) and control antibodies (IgG) together with purified GSC#4-secretd EVs. Vascular permeability was measured using in vivo Miles assay (n = 5). **Po0.01; ***Po0.001. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Permeability, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Control, Staining, Microscopy, Western Blot, In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Transfection, Blocking Assay, Binding Assay, Concentration Assay

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 3. Tumour-derived EV-transported Sema3A enhances vascular permeability. (a–c) EVs were purified from conditioned medium of GSC#4 that received either non-silencing RNA (siC) or Sema3A-targeting (siS3A), and applied to the left hemisphere of mouse brain. Vehicle (PBS) served as a control. (a) Evans blue extravasation is shown in OCT-included brains. Arrow indicated the site of EV dropping (left hemisphere). (b) Intensity of Evans blue extravasation was quantified in left and right hemispheres, including all brain areas (whole), only cerebrum (crbum) or cerebellum (crbellum). *Po0.05; **Po0.01; ***Po0.001. (c) Confocal analysis of the red blood cell marker Ter119 (red), Claudin5 (Cldn5, green) and Evans Blue (grey) in frozen brain sections. Nucleic acid staining is shown in blue (DAPI). Scale bar: 40 μm. Graph shows the quantification of Ter119 signal intensity from confocal analysis. **Po0.01. (d) Frozen tissue sections from subcutaneous ectopic and intracranial orthotopic GSC#9 xenografts were stained with Pecam (green) and Ter119 (red). Nucleic acid staining is shown in blue (DAPI). Scale bar: 20 μm. (e) Circulating blood EVs were purified from non-injected (non-tum EV, blue) and GSC-xenografted (xeno EV, red) mice and were analysed by flow cytometry for mouse (m) and human (h) CD63. Respective isotype controls (IgG) are shown in grey. Percentage represents the number of positive events for hCD63. (f) Human Sema3A (hSema3A) concentrations were measured by ELISA in circulating EVs purified from sera of non-injected ( −, light orange) and ectopically (ecto.) or orthotopically (ortho.) GSC xenografted (GSC, orange) mice. *Po0.05; **Po0.01. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Derivative Assay, Permeability, Control, Marker, Staining, Injection, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 4. Peripheral blood-isolated EVs from glioblastoma patients promote vascular permeability through a Sema3A/NRP1-dependent mechanism. Circulating EVs were purified from healthy donor and GBM patient sera. (a) Representative FSC/SSC dot-plot of the EV population, resuspended in filtered PBS, and analysed by flow cytometry together with 10-μm-calibrated beads (cal). EVs were further analysed by flow cytometry for Annexin V (AnnV, blue) and CD63 (blue) relative to their respective controls (Ctl, red). (b) Evans blue dye extravasation (Miles assay) of intradermic injected mice with purified EVs from healthy donors (Healthy, green) or GBM patients (GBM, orange). *Po0.05. (c) Intensity of Evans blue extravasation was quantified in left and right hemispheres, including all brain areas (whole), only cerebrum (crbum) or cerebellum (crbellum). **Po0.01; ***Po0.001. (d) Sema3A concentrations were measured by ELISA in purified-EV from healthy donors (green, n = 15) or GBM patient sera (red, n = 4). ***Po0.001. (e) Mice received intradermic injections of blocking (aNRP1A and aNRP1B) and control antibodies (IgG) together with purified GBM patient sera-derived EVs (GBM-EVs). Vascular permeability was measured using in vivo Miles assay. **Po0.01; ***Po0.001. (f) Graphs show the quantification of Evans blue signal intensity from confocal analysis of mouse epidermis after Miles assay. *Po0.05; **Po0.01. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Isolation, Permeability, Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Derivative Assay, In Vivo