Journal: iScience

Article Title: The atypical CDK activator RingoA/Spy1 regulates exit from quiescence in neural stem cells

doi: 10.1016/j.isci.2023.106202

Figure Lengend Snippet:

Article Snippet: CD9-Vio770 (clone, rat MZ3) , Miltenyi , Cat#130-102-384; RRID: AB_2659590.

Techniques: Luciferase, Recombinant, Reverse Transcription, Flow Cytometry, Plasmid Preparation, Software, High Content Screening

Journal: Journal of Hepatocellular Carcinoma

Article Title: A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis

doi: 10.2147/JHC.S423043

Figure Lengend Snippet: Purification of extracellular vesicles (EVs) by size exclusion chromatography preserves immunoreactivity assessed by fluorescent-nanoparticle tracking analysis (F-NTA). ( A ). Shows effectiveness of purification after using lipophilic membrane dye (Cell Mask Plasma Membrane Stain) for staining EV’s. According to percentages of the positive stained vesicles, most of the EV’s are membranous. ( B ). A pie chart shows the EV population diversity of exosome and microvesicle after immunocaptured using PE-labeled CD9 antibody and PE-labeled ARF6 antibody from cell culture, and clinical samples (normal serum, cirrhosis with or without HCC). As predicted, the F-NTA analysis shows exosome release increased in chronic liver disease as compared to only among healthy individuals. A large percentage of EVs remain unlabeled. ( C ). Show clinical application of F-NTA to quantify liver-derived EVs using PE-labeled antibodies to ASGPR1. The percentage of liver-derived vesicles was lower in cirrhosis and HCC groups than the healthy controls.

Article Snippet: PE-labeled monoclonal rabbit and mouse antibodies against GPC3 (sc-390587 PE), AFP (sc-8399 PE), CD9 (sc-13118 PE), ARF6 (sc-7971 PE), and ASGPR1 (sc-166633 PE) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Purification, Size-exclusion Chromatography, Membrane, Clinical Proteomics, Staining, Labeling, Cell Culture, Derivative Assay

Journal: Journal of Hepatocellular Carcinoma

Article Title: A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis

doi: 10.2147/JHC.S423043

Figure Lengend Snippet: Assay calibration using exosome purified from Huh7 culture. Approximately 1×10 7 Huh 7 cells were cultured in a 10-cm tissue culture plate. Media were collected after 72 hours and exosomes were precipitated from 1-mL cell-free supernatants by precipitation. Exosomes were recovered after centrifugation. The exosome pellet was resuspended in 100μL of PBS with 1%BSA and incubated with PE-labeled GPC3, AFP, and CD9 overnight. The next day, samples were diluted in 1 mL water and passed through a column. Extracellular vesicles (EVs) collected from flow-through were diluted in water (1:10) and immediately analyzed by fluorescent-nanoparticle tracking analysis (F-NTA) using light scattered mode (LSM) and fluorescence mode (FM). ( A ). Video images of PE-conjugated CD9 antibody labeled EVs were captured from Huh7 and HuCCA-1 culture and measured by F-NTA in LSM and FM. ( B ). Representative video image of PE-labeled GPC3 antibody-labeled HCC exosomes captured from HuCCA-1 and Huh7 cells and measured by NTA in LSM and FM. ( C ). Total exosomes and HCC exosomes are released per tumor cell. The total exosome release from HuCCA-1 cells is more than from Huh7 cells. HuCCA-1 cells release a minimum number of HCC exosomes as compared to Huh7 cells. Statistical significance levels were presented as ns for non-significant, * for p<0.05, ** for p<0.01.

Article Snippet: PE-labeled monoclonal rabbit and mouse antibodies against GPC3 (sc-390587 PE), AFP (sc-8399 PE), CD9 (sc-13118 PE), ARF6 (sc-7971 PE), and ASGPR1 (sc-166633 PE) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Purification, Cell Culture, Centrifugation, Incubation, Labeling, Fluorescence

Journal: Scientific reports

Article Title: Evaluation of circulating small extracellular vesicle-derived miRNAs as diagnostic biomarkers for differentiating between different pathological types of early lung cancer.

doi: 10.1038/s41598-022-22194-0

Figure Lengend Snippet: Figure 2. Isolated sEV-enriched fractions from participants’ plasma (a–c) TEM images showing that sEVs of three groups are oval or bowl-shaped capsules without a nucleus. d. The Western blot analysis of sEVs, markers CD9, CD63and TSG101were all detected in the sEV-enriched fractions isolated from plasma, and Calnexin, a negative marker of sEVs was absent in our isolated sEVs-enriched fraction samples. (e–g) NTA showed that the diameter and concentration of EVs for three groups. CL, control cell lysate.

Article Snippet: Small EV-enriched supernatant was denatured in 5 × sodium dodecyl sulfonate (SDS) buffer and subjected to Western blotting (10% SDS–polyacrylamide gel electrophoresis; 50 μg protein/ lane) using rabbit polyclonal antibodies against CD63 (sc-5275, Santa Cruz, CA, USA), CD9 (60232-I-Ig, Proteintech, Rosemont, IL), TSG101 (sc-13611, Santa Cruz) and calnexin (10,427–2-AP, Promega, Madison, WI).

Techniques: Isolation, Clinical Proteomics, Capsules, Western Blot, Marker, Concentration Assay, Control

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