Studies on the isolation,purification,specific molecule expression and phagocytosis of human microglia
- VernacularTitle:人小胶质细胞的分离、纯化、特异分子表达与吞噬功能研究
- Author:
Xiuyan HUANG
;
Yaoying ZENG
;
Cai ZHANG
;
Bin ZHU
- Publication Type:Journal Article
- Keywords:
Microglia;
CD68;
Phagocytosis
- From:
Chinese Journal of Pathophysiology
1989;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To explore a method of isolation,purification,culture and identification of human microglia.METHODS:The brain tissue from abortive fetus was sterilely obtained,then chopped and triturated gently.The suspension was filtered and centrifuged to separate and isolate mixed glia cells.The cell density was adjusted to 2?1010 cells/L with DMEM/F12 complete medium and cultured in 75 cm2 flask at 37 ℃ in CO2 incubator(marked as first 0 d).After 7-10 days culture,floating cells including microglia and oligodendrocytes appeared in the flask.The first time purification was carried out to obtain highly purified microglia,for which the flasks were shaken gently.The floating cells were collected and transferred into a new flask(marked as second 0 d).4-5 days later,when microglia and oligodendrocytes grew in confluent state,the second purification was performed,for which the mixed cultured cells were digested with trypsin-EDTA and cell suspension was centrifuged,then the cell pellet was suspended by DMEM/F12 complete medium and cultured in flask(marked as 0 h).After 2 h,non-microglial cells including oligodendrocytes and cell debris were removed,and fresh medium was added into the adherent cells.In this way,the whole procedure of microglia purification was completed and the highly purified microglial cells were obtained.After purification,the expression rate of CD45,CD11b and CD68 on/in microglia were detected by laser-confocal microscopy and flow cytometry on the basis of immuno-fluorescence staining to identify the purity of microglial cells.Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres.The level of activation was identified by the phenomena of membrane ruffling in combination with fluorescent phalloidin staining.RESULTS:More than 98% of the microglias that we isolated and purified expressed CD45,CD11b and CD68.Almost all of the microglias could phagocytize fluorescent microbeads CONCLUSION:We succeed in isolating and purifying human microglias,which express CD45,CD11b and CD68.Almost all of the cells exhibit phagocytic function.So these cells will be useful for further functional and proteomic studies.
