Isolation,extraction and identification of exosomes from non-small cell lung cancer A549 cell culture supernatant / 中国生物制品学杂志

ABSTRACT

@#Objective To isolate,extract and identify exosomes from the culture supernatant of non-small cell lung cancer A549 cells. Methods Exosomes from supernatant of non-small cell lung cancer A549 cells were isolated by ultracentrifugation(Ult)and modified method[Ult combined with Exo Quick~(TM)(Ult-Exo Quick~(TM),U-EQ)],which were determined for the concentration by BCA,measured for the particle size distribution by nanoparticle tracking analysis(NTA),observed for the morphology by fluorescence staining and transmission electron microscope(TEM),and detected for the expression of CD63 and HSP70 proteins on the surface of exosomes by Western blot. Results The concentrations of exosomes isolated by U-EQ and Ult were 1. 355 and 0. 909 mg/mL,respectively;The total number of particles was 4. 65 × 10~9 and 4. 06 × 10~9 particles/mL,the peak sizes ranged from 81. 5 to 165. 5 and 59. 5 to 176. 5 nm,and the average size was(152. 3 ± 8. 8)and(94. 3 ±9. 8)nm,respectively;The exosomes were all 30～150 nm in diameter,while the morphology of exosomes extracted by U-EQ method was more uniform;CD63 and HSP70 proteins were expressed in all of them,while the exosomes extracted by U-EQ method showed higher expression. Conclusion Both U-EQ and Ult methods can isolate exosomes from the supernatant of non-small cell lung cancer A549 cells,while the concentration of exosomes extracted by U-EQ method is higher.