Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Development of MMR Slurry for high purity isolation of extracellular vesicles from human CSF and plasma. A. Overview of approach. Immuno-isolation of neuron-specific EVs from human CSF or plasma for interrogation of neuron-derived RNA or protein cargo. B. Principle underlying MMR Slurry technique. Capto Core 700 beads have pores allowing biomolecules less than 700 kDa to enter, and once free proteins enter, they stay in the beads, which can be removed to leave pure EVs. C. EV isolation workflow with MMR Slurry. Samples are mixed with Capto Core beads and then beads are pelleted and discarded. D. Western blot of CD9, CD63, CD81, and albumin of human CSF after MMR Slurry purification with increasing amounts of Capto Core beads shows a strong enrichment of tetraspanins relative to albumin. E. Total protein stain of CSF after MMR Slurry purification with increasing amounts of Capto Core beads. F. Western blot of L1CAM in CSF after MMR Slurry purification with increasing amounts of Capto Core beads. G. Western blot of CD9, CD63, CD81, and albumin after SEC of plasma followed by MMR Slurry purification with increasing amounts of Capto Core beads shows a strong enrichment of tetraspanins relative to albumin. H. Total protein stain after size exclusion chromatography of plasma, followed by MMR Slurry purification with increasing amounts of Capto Core beads. Ratio of 50% Capto Core (CC) bead slurry to protein is indicated in μL CC Slurry/μg protein in sample. For D-H, equal fraction each sample was loaded onto each gel. I. Simoa quantification of EV purity comparing MMR Slurry after SEC or SEC from plasma. Levels of CD9, CD63, and CD81, and albumin were measured between conditions (based on two replicates for each condition and Simoa measurements made in duplicate for each replicate). Ratios of CD9, CD63, and CD81 were averaged to calculate relative EV yield and divided by relative ratios of albumin to calculate EV purity. EV purity of SEC is set to 1. Two separate replicates of all samples were used and Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following primary antibodies were used for Western blot at the corresponding dilutions: M38 for CD81 (Thermo Fisher Scientific) at 1:666, H5C6 for CD63 (BD) at 1:1000, CD9 (Millipore) at 1:1000, EPR18998 for L1CAM (Abcam) at 1:500, ab47441 for GJA1 at 1:500, F-10 for albumin (Santa Cruz) at 1:1000, E7K3G (Cell Signaling Technology) at 1:5000 or AF5269 (R&D Systems) at 1:1000 for NRXN3.

Techniques: Isolation, Derivative Assay, Western Blot, Purification, Staining, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Development and validation of EV immuno-isolation method and application to NRXN3. A. Schematic of immuno-isolation of EVs enriched by ultracentrifugation from the cell culture media of K562 cells. B. Western blotting of CD81 after immuno-isolation of EVs. Equal amounts of EVs obtained by differential ultracentrifugation of K562 cell culture media were loaded as input or immuno-isolated using either an anti-CD81 antibody or a control non-specific antibody. PD = pulldown, FT = flow-through C. Schematic of EV immuno-isolation experiment from human plasma or CSF for western blotting analysis. EVs are immuno-isolated from biofluid without initial EV purification. Immuno-isolation was performed using a target antibody or control antibody (PD1 = pulldown 1) and the flow-through is subjected to a second immuno-isolation with the target antibody (PD2 = pulldown. 2) D. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human CSF. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. E. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human plasma. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. F. Schematic of EV immuno-isolation experiment from human plasma or EVs enriched by SEC from human plasma for assessment by Simoa in the flow-through (FT). G. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of human plasma after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). H. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of EVs enriched from human plasma by SEC after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). For G and H, two separate replicates of all samples were used and Simoa measurements were taken in duplicate for each sample. Error bars indicate SD. I. Schematic of NRXN3 immuno-isolation experiment. NRXN3 in pulldown fraction (PD) was measured using Western blotting. NRXN3 in flow-through (FT) was measured using Simoa. Immuno-isolation experiment was performed using antibody against target (NRXN3) or control (GFP or isotype control) J. Western blotting of NRXN3 after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). K. Western blotting of NRXN3 after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). For J and K, Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following primary antibodies were used for Western blot at the corresponding dilutions: M38 for CD81 (Thermo Fisher Scientific) at 1:666, H5C6 for CD63 (BD) at 1:1000, CD9 (Millipore) at 1:1000, EPR18998 for L1CAM (Abcam) at 1:500, ab47441 for GJA1 at 1:500, F-10 for albumin (Santa Cruz) at 1:1000, E7K3G (Cell Signaling Technology) at 1:5000 or AF5269 (R&D Systems) at 1:1000 for NRXN3.

Techniques: Isolation, Cell Culture, Western Blot, Purification

Journal: Nanomaterials

Article Title: Cytochalasin B Treatment and Osmotic Pressure Enhance the Production of Extracellular Vesicles (EVs) with Improved Drug Loading Capacity

doi: 10.3390/nano12010003

Figure Lengend Snippet: The proteins expressions, cellular uptake, and dendrimer loading capability of hypo-CIMVs compared to naturally-secreted EVs, hypo-EVs, and CIMVs: ( A ) Amount of total vesicular protein ( n = 3); ( B ) Expression of transmembrane protein CD81 compared to intravesicular protein MIF; ( C ) Immunogold TEM images for assessing the CD63 (transmembrane marker) expression; ( D ) Cellular uptake of the produced EVs; ( E – G ) Loading of ( E ) G7-NH2, ( F ) G7-COOH, and ( G ) G7-Ac to the produced EVs. Significance levels are indicated as # p < 0.10, * p < 0.05, and ** p < 0.01, compared to the EVs obtained from non-CB treated cells. The dendrimer loading to the EVs was analyzed from at least three independent experiments ( n ≥ 3).

Article Snippet: The blot was blocked with 5% skim milk for 1 h followed by overnight incubation in primary antibody against CD81 (monoclonal anti-CD81 SC-166028, Santa Cruz Biotechnology, San Jose, CA, USA) and macrophage migration inhibitory factor (MIF) (monoclonal anti-MIF, MAB289, R&D Systems) at 4 °C.

Techniques: Expressing, Marker, Produced

Journal: mBio

Article Title: Hepatitis C virus infects and perturbs liver stem cells

doi: 10.1128/mbio.01318-23

Figure Lengend Snippet: Liver organoids grow from HCV-infected individuals and show similar differentiation potential. ( A ) Representative brightfield microscopy images of liver organoids grown from uninfected (NV) or HCV + donors are shown in the stem cell (EM) and differentiated (DM) states. Organoids are morphologically distinct in EM vs DM states, but each state was morphologically identical across all six NV and HCV + donors. ( B ) Quantitative PCR (qPCR) quantification of hepatocyte stem cell marker LGR5 and hepatocyte markers ALB, CYP3A4, and CYP2B6 in DM organoids from three NV donors and three HCV + donors, and in two primary hepatocyte samples relative to EM. For each gene, data were pooled from n ≥ 2 biological replicates per organoid or hepatocyte donor and represented as mean ± SD. Transcript expression was normalized to 18S and plotted as a fold change over the gene’s expression in EM (ΔΔC T ) which was set to one and is marked by a dotted line. Fold change was plotted on a log10 scale. ( C ) qPCR quantification of HCV entry markers, CD81, OCLN, CLDN1, and SR-B1 in the same samples as in ( B ). EM expression levels were set to one (marked by a dotted line), and fold change in DM was plotted on a linear scale. ( D ) Representative light-sheet microscopy images are shown for differentiated liver organoids (DM) stained for hepatocyte markers HNF4α and ALB, HCV entry factors CLDN1 and CD81, or apical membrane marker ZO1.

Article Snippet: Primary antibodies we used include CD81 (BD Pharmigen, JS-81), claudin-1 (Thermo Scientific, 2H10D10), HNF4α (Cell Signaling Technology, C11F12), albumin (Sigma-Aldrich, HSA-11), and ZO1 (Thermo Scientific, 1A12).

Techniques: Infection, Microscopy, Real-time Polymerase Chain Reaction, Marker, Expressing, Staining, Membrane

Journal: Oncotarget

Article Title: Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel

doi:

Figure Lengend Snippet: (A) Exosomes isolated from DU145 Tax-Sen and DU145 Tax-Res cells were visualized by transmission electron microscopy (bar size: 100 nm); (B) Nanoparticle tracking analysis on an LM10 Nanosight demonstrating a mean size of 100 nm for DU145 Tax-Res and 120 nm for DU145 Tax-Sen exosomes. The size distribution and relative concentration were calculated by the Nanosight software ( n = 3); (C) Western blot analysis of 10 μg lysates from DU145 Tax-Sen and DU145 Tax-Res cells and exosomes and probed for the indicated proteins ( n = 2); (D) Flow cytometric analysis of the mean fluorescence intensity (MFI) for a panel of exosomal markers CD9, CD63, CD81 and CD82. Data is presented as means of triplicate experiments.

Article Snippet: The primary antibodies used in this study against Rab 5, CD81, CD82, Alix, Emmprin, Caveolin-1 were obtained from Cell Signaling Technology; TSG101, CD9 and CD82 from Abcam, MDR-1 and MDR-3 from Gene Tex; AIF, CD9, Endophilin A2 and CD63 from Santa Cruz Biotechnology; PACSIN2 from Abcam and Santa Cruz Biotechnology; Cyp17A from Novus Biologicals; IgG1-APC from Biolegend.

Techniques: Isolation, Transmission Assay, Electron Microscopy, Concentration Assay, Software, Western Blot, Fluorescence

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 1. Characterization of miR-185 enriched MSC-EVs. (A) Isolated miR-185 MSC-EVs were characterized by TEM. Representative image is shown (scale bar ¼ 50 nm). (B) NTA was performed on the EVs to determine their concentration and size. EV diameter was measured and represented as mean ± SD, n ¼ 3 independent experiments performed in triplicate. Western blotting (insets) for EV markers CD9, CD81 and flotillin-1. (C) qPCR analysis for expression of miR-185 in EVs. n ¼ 5 inde- pendent experiments performed in triplicate (p < .01 versus Ctrl).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Isolation, Concentration Assay, Western Blot, Expressing