[Establishment of a method for separating macrophage migrasomes]
Yongbin Ma1, Leyu Zhao2, Dan Zhou1
1Department of Central Laboratory, Jintan Hospital, Jiangsu University, Jintan 213200, China.
Abstract:
Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.
Insights
Researchers developed an efficient method to isolate high-quality migrasomes from macrophages. This technique allows for detailed analysis of migrasome structure and RNA content, advancing cell biology research.
Area of Science:
- Cell Biology
- Macrophage Biology
- Extracellular Vesicles
Context:
- Migrasomes are novel cellular structures originating from macrophages.
- Characterizing migrasomes is crucial for understanding intercellular communication.
- Existing methods for migrasome isolation are limited.
Purpose:
- To establish an efficient and reliable method for isolating migrasomes from RAW264.7 macrophages.
- To characterize the morphology and molecular markers of isolated migrasomes.
- To analyze the RNA content within purified migrasomes.
Summary:
- A novel method using 0.45 μm filter reverse filtration and elution was developed for migrasome isolation from RAW264.7 cells.
- Scanning and transmission electron microscopy confirmed the oval, vesicle-like structure of isolated migrasomes.
- Western blot analysis identified specific migrasome markers (PIGK, EOGT, TSPAN4) and excluded common extracellular vesicle markers (TSG101, ALIX).
- Migrasomes were found to contain small RNA fragments (25-200 nt).
Impact:
- This study provides a robust protocol for obtaining high-purity migrasomes.
- The established method facilitates further investigation into migrasome function and biogenesis.
- Findings contribute to the understanding of macrophage-derived extracellular components and their roles in biological processes.
