Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: (A) Workflow for collection of extracellular vesicles from RAW264.7 macrophages infected with L. donovani parasites. Infection was performed in media supplemented with exosome-depleted serum. After 24 h, the culture medium was removed. Cultures were washed to remove uninternalized parasites and replenished with fresh medium supplemented with exosome-depleted serum. After an additional 48 h, culture medium was recovered, pooled, and processed following the centrifugation and filtration steps shown in the figure. (B) Nanoparticle tracking analysis was performed from which particle size distribution and particle concentration was obtained. Plot of particles/cell was calculated using cell count at the end of the infection. Data for graphs were obtained from multiple experiments (ceEV n = 7, LieEV n = 7; * P = 0.0082). (C) Representative image of vesicles in LieEV preparation processed for scanning electron microscopy. (D) Representative transmission electron microscopy image of immunogold CD9-labeled particles in LieEVs. Arrows point to gold particles denoting reactivity of antibody.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Centrifugation, Filtration, Concentration Assay, Cell Counting, Electron Microscopy, Transmission Assay, Labeling

Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: The proteome of LieEVs recovered after 72-h infection was compared with the proteome of ceEVs from uninfected cells. (A) Venn diagram was plotted using proteins identified by mass spectrometry and partitioned according to sample type. The presence and absence of host molecules with and without infection and common to both samples are indicated. (B) The protein content of the preparations was revealed by Ponceau S staining of the blots to normalize for material loaded into each well for each sample type. Approximately 1 × 10 10 particles from EV preparations from three replicate experiments and 50 μg of lysates from infected cells at the 72 h infection point were analyzed. (C) Western blot was performed to confirm the presence of known and novel exosome markers. Uninfected macrophages were treated in an identical manner as infected samples. The blots were then probed with anti-CD9, stripped, and probed with anti-Annexin A3. Identical blots were probed initially with anti-CD63, stripped, and probed with anti-calnexin. (D) Quantification was performed by measuring the mean gray background area using ImageJ software. Background pixel density was subtracted from the inverse of each measurement to obtain relative quantification values. Analysis of the blots showed that CD9 was significantly more abundant in LieEVs than ceEVs and cell lysates (* P = 0.0261, n = 3), whereas levels of CD63 were comparable for all samples. Annexin A3 was significantly more abundant in LieEVs than in ceEVs (* P = 0.0123, n = 3). Calnexin was barely detected in EVs as compared with cell lysates. Statistical test for differences was by ANOVA. Source data are available for this figure.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Mass Spectrometry, Staining, Western Blot, Software, Quantitative Proteomics

Journal: bioRxiv

Article Title: Orally Delivered Milk-Derived Nanovesicles Loaded with Connexin 43 Peptides for Targeted Cardiac Ischemia-Reperfusion Therapy

doi: 10.1101/2025.01.01.630994

Figure Lengend Snippet: A) Schematic of mEV isolation protocol showing the steps of the procedure. Fractions referred to are those collected during Sepharose Column (SEC) fractionation. Fractions 7 through 9 (F7-F9) show highest levels of mEV enrichment and samples used in this study were pooled from F7-F9. Fractions >14 show enrichment for contaminating milk proteins such as casein, with low or no evidence of mEVs, and are typically discarded. B) Single Molecule Localization Microscopy (SMLM) image of an isolated mEV isolated by the protocol in (A) tagged for CD81 (red) and CD63 (teal). C) Negative stain transmission electron microscopy (TEM) indicating dense accumulations of mEVs generated by the isolation protocol. D) Calcein AM loading assay illustrating membrane intactness of isolated mEVs E) Western blotting of mEV isolates for CD81, CD9, TSG-101, and negativity and markedly reduced expression of Calnexin and Casein, respectively. Lysates from HeLa cells are included as a control. Sepharose fraction 16 (F16) shows elevated casein, but no evidence of EV markers. F) Nanoparticle Tracking Analysis (NTA) of an mEV isolate indicating >5×10 12 mEVs per mL, with average nanoparticle size of 125.0 nm, consistent with small EVs. G) SMLM-generated size distribution of CD9/CD81/CD63-positive mEVs, showing a similar histogram morphology to NTA results in panel F.

Article Snippet: Overnight primary antibody incubation was performed and primary antibodies were diluted in the blocking buffer as follows: CD81 (Cell Signaling Technology, 56039S, 1:1000) CD9 (Novus Biologicals, NB500-494, 1:1000), Calnexin (MilliporeSigma, AB2301, 1:5000), TSG101 (Bethyl Laboratories Inc. A303-506A, 1:5000), Casein (Abcam, Ab166596, 1:2000), Cx43 Cytoplasmic-Loop (Invitrogen, PA5-11632, 1:1000), and antibodies against the Cx43 CT (MilliporeSigma, C6219, 1:4000 and Abcam, Ab87645, 1:1000).

Techniques: Isolation, Fractionation, Microscopy, Staining, Transmission Assay, Electron Microscopy, Generated, Membrane, Western Blot, Expressing, Control

Journal: Physiological reports

Article Title: Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.

doi: 10.14814/phy2.16016

Figure Lengend Snippet: FIGURE 3 EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright-field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle-derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three-way ANOVA.

Article Snippet: Samples were then stained with the following antibodies and dilutions: anti- human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti- human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti- human thrombospondin (THSD- 1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO).

Techniques: Marker, Expressing, Imaging, Flow Cytometry, Derivative Assay

Journal: Transfusion Medicine and Hemotherapy

Article Title: Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation

doi: 10.1159/000508712

Figure Lengend Snippet: FACS-based characterization of isolated EVs (second round). A FACS histograms depicting the relative fluorescence/marker intensity of EV preparation 2.1 (black line) against unstained EV particle control (grey line). B Corresponding marker expression in HCT116 cells (extracellular staining for CD9, CD63, and CD81 and intracellular staining for Alix, TSG101, and calnexin). C, E Mean fluorescence intensity (MFI) raw values of CD63 (C) and CD81 (E) marker expression from laboratories 2.1–2.4. D, F MFI values per particle concentration of CD63 (D) and CD81 (F; left y axis) against the respective particle concentration per ml CCM (right y axis).

Article Snippet: HCT116-derived EVs were captured on anti-human CD9 beads for flow detection (Exosome-Human CD9 beads; Thermo Fisher Scientific).

Techniques: Isolation, Fluorescence, Marker, Control, Expressing, Staining, Concentration Assay