[Isolation of exosomes derived from dendritic cells by ultrafiltration centrifugalization and their morphologic characteristics].
OBJECTIVE
To investigate a method for isolating exosomes by ultrafiltration centrifugalization based on their physical size and density so as to advance traditional method and promote recovery rate.


METHODS
The culture supernatant of murine dendritic cells line (DC2.4) was collected and clarified through a hollow fiber cartridge to remove cells and their debris. The clarified supernatant was concentrated by Amicon ultrafiltration tube and then the concentrated supernatant was purified when loaded into density gradient centrifugation to obtain the DC-Ex. The traditional differential centrifugation method was used in control group. Transmission electron microscope (TEM) was used to identify the morphology of the two products. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were employed to analyze their proteins.


RESULTS
The obtained pellets possessed the features of dendritic cell exosomes described in previous literature, including morphology and SDS-PAGE profile. Moreover,these particles contained characteristic molecular markers MHC class I, MHC class II, CD80 and CD86. Compared with the differential centrifugation method, ultrafiltration centrifugalization technique is a shorter and more producible process resulting in production being highly purified.


CONCLUSIONS
Ultrafiltration centrifugalization technique is a convenient and useful method for DC-Ex isolation.
Center of Diagnosis and Treatment for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China. freshmanww@163.com
Wei
W
Wang
Jian-Qi
JQ
Lian
Ping-Zhong
PZ
Wang
Lei
L
Pan
Xin-Ying
XY
Ji
Xue-Fan
XF
Bai
Zhan-Sheng
ZS
Jia
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002498:Centrifugation / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology* / Q000648:ultrastructure; D055354:Exosomes / Q000648:ultrastructure*; D051379:Mice; D008854:Microscopy, Electron; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods*
D011506:Proteins