Isolation and identification of exosomes from mouse primary peritoneal macrophages
Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3％mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101)by Western blot.Results About 5 × 106 peritoneal macrophages with the purity of(99.17±0.65)％were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucer-like vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of 175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary perito-neal macrophages.ExoQuick TC exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.
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