A novel method for successive acquisition of high purity and yield of microglia
10.3760/cma.j.issn.1671-8925.2011.04.015
- VernacularTitle:一种连续获取大量高纯度小胶质细胞的培养方法
- Author:
Ye-Hai LI
1
;
Kun QIN
;
Zhi-Lin LANG
;
Ling-Sha QIN
;
Yu-Xi ZOU
;
Xiao-Dan JIANG
Author Information
1. 南方医科大学珠江医院
- Keywords:
Microglia;
Cell culture technology;
Laboratory technique and method
- From:
Chinese Journal of Neuromedicine
2011;10(4):382-388
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an easy culture method of successively getting high purity and yield of microglia. Methods Cortices of neonatal Wistar rats (1-3 days old) were employed in this experiment. The first-generation microglial cells were isolated from the mixed glial culture by mechanical means (gently shaking and blowing with pipette). After the mixed glial cells being passaged at a density third generations ofmicroglial cells were harvested. CD1 lb/c, CD45, CD80, CD86 and GFAP were employed as the identification markers in detecting the phenotypes and purity of different generation of microglial cells by scanning electron microscope and flow cytometry. Immunofluorescence staining and CCk8 vitality measurement were used to judge the expression of CD11b/c and detect the proliferation of microglia cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. Results High yield and purity of microglial cells were stably obtained in this experiment. CD11b/c, CD45, CD80 and CD86 positive expressions were noted in the first and third generations of microglial cells by flow cytometry; CD1 1b/c positive expression was noted in the first,second and third generations of microglial cells by immunofluorescence staining. No obvious differences in the 3 different generations of microglia cells were found on proliferation ability by CCk8 vitality measurement, and on morphology and phenotypes by scanning electron microscope; no obvious differences in the first and third generations of microglia cells were found on phagocytic ability (P＞0.05).Conclusion High yield and purity of microglial cells can successively obtain through the above method;no significant differences are noted among different generations of microglia cells on purity, morphology,phenotypes, proliferation activity and phagocytic ability.
