Exosomes are small vesicles with a diameter of about 30-150 nm secreted by living cells. They have a typical lipid bilayer structure. They participate in the communication between cells, the transport of substances, and the maintenance of normal physiological processes. They are also related to the occurrence of diseases. Exosome endocytosis tracing is to label exosomes with tracers (such as organic dyes, fluorescent proteins, contrast agents, radionuclides, etc.) and track exosomes in animals with specific imaging methods, so as to observe the biological behavior of exosomes in vivo and detect the endocytosis of exosomes by target cells, and to facilitate further research on the function of exosomes. At present, the commonly used labeling method for exosomes is lipophilic dyes, and the commonly used imaging method is optical imaging.
1. Experimental principle
Lipophilic dyes used for exosome labeling can stably bind to the lipid region of the cell membrane and emit fluorescence. Among them, the commonly used membrane protein fluorescent dyes are PKH67 (green)/PKH26 (red). After incubation with isolated exosomes, the exosomes can be labeled and made fluorescent. By adding fluorescently labeled exosomes and incubating with target cells, the fluorescence enrichment in the target cells is detected by fluorescence microscopy to determine whether the target cells can internalize the exosomes. Optical imaging in imaging is divided into bioluminescence imaging and fluorescence imaging. Among them, bioluminescence imaging is a chemiluminescence imaging method produced by the catalysis of artificially injected luciferin substrates by self-synthesized luciferase, which requires an ultra-sensitive camera to detect. Fluorescence imaging uses organic dyes or fluorescent proteins to emit signals under the stimulation of external light sources to form images.
2. Experimental steps
2.1 Lipophilic dye labeling experiment
2.1.1 Exosome protein quantification: Take an appropriate amount of exosomes for BCA concentration determination to determine the amount of exosome protein.
2.1.2 Preparation of dye working solution: Dilute the PKH67/PKH26 linker storage dilution 10 times with Diluent C to prepare a dye working solution with a concentration of 100 μM.
2.1.3 Exosome staining: Add dye working solution to exosomes (it is recommended to add 50 μL of dye working solution to 10-200 μg of exosome protein and 50 μL of dye working solution to 10-200 μg of exosome protein). After adding the dye working solution, mix it by vortexing for 1 min, and then incubate it for 10 min. Add 10 mL of 1 × PBS to the incubated exosome-dye complex and mix it. Extract the exosomes again according to the exosome extraction method to remove excess dye, and finally take 200 μL of 1 × PBS to resuspend the precipitate. The precipitate is the fluorescently labeled exosome.
2.1.4 Add fluorescently labeled exosomes and incubate with target cells. The incubation time depends on the experiment.
2.1.5 Observe the fluorescence enrichment under a fluorescence microscope.
2.2 The experimental principle and steps of fluorescence imaging are the same as those of dye labeling, but they are different in terms of fluorescence quantization, color and half-life. The emission wavelength range of organic dyes is 502 to 734 nm, which provides a variety of options for selecting suitable probes for exosome labeling.
2.3 Bioluminescence imaging: According to the experimental needs, cells labeled with luciferase are inoculated by tail vein injection, subcutaneous transplantation, or in situ transplantation, and substrates are injected at different times to excite luminescence to display cell-derived exosomes for imaging.
3. Example of results
L6 cells were taken up by PKH67-labeled exosomes (green) after co-culture. PKH67-labeled exosomes were localized in the nucleus of L6 cells.
References
[1] Xiuhui,Wang,Zhuokai,Li,Yin,Cui.Exosomes Isolated From Bone Marrow Mesenchymal Stem Cells Exert a Protective Effect on Osteoarthritis via lncRNA LYRM4-AS1-GRPR-miR-6515-5p.9,(2021)
[2] Zeng Li a,Ying jie Wang, Shuai Xiang, Zhibo Zheng.Chondrocytes-derived exosomal miR-8485 regulated the Wnt/bcatenin pathways to promote chondrogenic differentiation of BMSCs.12,065,(2019)
[3] Qiang Zhou, Hongchang Yang, Hongchi Pan.Exosomes isolated from the miR-215-modified bone marrow mesenchymal stem cells protect H2O2-induced rat myoblasts via the miR-215/ FABP3 pathway.119,(2021)