Quantifying Exosome Characteristics and Internalization

The exosome size was determined using a dynamic light scattering system (Otsuka ELS-Z, Japan). Exosomes isolated from the plasma of WT and CrebH^−/− mice were quantified using an ExoELISA-ULTRA assay kit (SBI System Biosciences) according to the manufacturer’s guidelines [28 (link)]. Exosomal markers were estimated by immunoblotting using antibodies against TSG101 (Abcam), CD9 (Abcam), and Histone H3 (Sigma-Aldrich). Exosomes were labeled using an ExoGlow-Membrane EV labeling kit (SBI System Biosciences) following the manufacturer’s instructions. Labeled exosomes were cocultured with bEnd.3 cell lines for the indicated time. Images were obtained using fluorescence microscopy (Olympus, Tokyo, Japan). Exosomal proteins were quantified using Bradford protein assay and bEnd.3 cells were treated with exosomes (30 µg/mL protein concentration contained in exosome) for 24 h.

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Lee S.H., Moon S.J., Woo S.H., Ahn G., Kim W.K., Lee C.H, & Hwang J.H. (2023). CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA. Cell & Bioscience, 13, 116.

Publication 2023

TSG101 Histone H3 ExoGlow-Membrane EV labeling kit fluorescence microscopy

Antibodies Assay Bend Cell lines Cells Exosome Fluorescence microscopy Histone h3 Membrane Mice Plasma Protein Tsg101

Corresponding Organization :

Other organizations : Korea Research Institute of Bioscience and Biotechnology, Daejeon University, Korea University of Science and Technology

Top 5 similar protocols

Variable analysis

independent variables

Genotype (WT vs. CrebH^-/-)

dependent variables

Exosome size
Exosome quantity
Exosomal protein markers (TSG101, CD9, Histone H3)
Exosome uptake by bEnd.3 cells
Protein quantification in bEnd.3 cells treated with exosomes

control variables

Dynamic light scattering system (Otsuka ELS-Z, Japan)
ExoELISA-ULTRA assay kit (SBI System Biosciences)
Antibodies for exosomal markers (TSG101, CD9, Histone H3)
ExoGlow-Membrane EV labeling kit (SBI System Biosciences)
BEnd.3 cell line
Exosome protein concentration (30 µg/mL)
Incubation time (24 h)

controls

Positive control: WT mouse plasma exosomes
Negative control: CrebH^-/- mouse plasma exosomes

Annotations

Based on most similar protocols

To optimize the exosome isolation, the protocols suggest using commercially available kits such as ExoQuick-TC (Protocol 1), Minute™ Hi-Efficiency Exosome Precipitation Reagent (Protocol 2), and Total Exosome Isolation Reagent (Protocol 4). These kits are designed to improve the efficiency and reproducibility of exosome isolation.

The protocols use various methods to characterize the isolated exosomes, including nanoparticle analysis (Protocols 1 and 3), transmission electron microscopy (Protocols 2, 3, and 5), and western blotting for exosomal markers such as CD9, CD63, and TSG101 (Protocols 1, 2, 3, and 5). These validation steps help confirm the accuracy and purity of the exosome preparations.

The input protocol and Protocol 4 both use a quantitative Bradford protein assay to determine the protein concentration of the isolated exosomes, which is important for ensuring consistent dosing during co-culture experiments.

Protocol 3 mentions the use of exosome-depleted FBS for in vitro experiments, which helps minimize the interference of serum-derived exosomes with the experimental system.

Several protocols (Protocols 1, 3, and 5) provide specific details on the centrifugation steps used for exosome isolation, including the g-forces and durations, which are critical for reproducibility.

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As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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