title: A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film.
abstract: Microglia are implicated in both neuroprotection and neurodegeneration, and are a key area of interest with respect to various CNS diseases. Until now, primary microglia prepared by various isolation methods have been widely used to investigate their role in CNS diseases. However, there are some problems with the current isolation methods, such as the numbers of animals required in order to obtain sufficient numbers of microglial cells due to low yields, and also the long periods of culture required. We herein describe a simple, high-yield method for isolating not only primary microglia, but also immortalized microglial cells. Our method allows for the isolation of an almost pure population of microglia with only two steps. First, a primary mixed neural culture was prepared from the brains of 3-day-old postnatal rats. Next, primary microglia were collected for 2 h by adhesion to Aclar plastic film. The average yield by this method was approximately 50 times higher than that of the conventional shaking method. Immortalized microglial cells could also be prepared based on this procedure. A plasmid vector encoding the SV40 large T antigen was transfected into the mixed neural culture using a calcium phosphate precipitation method. Then, proliferating immortalized microglia were collected after several weeks in a similar fashion. Several clones were obtained by limited dilution and one of the immortalized cell lines was designated SMK. The SMK cells exhibited markers specific for the microglia lineage, including Iba-1, CD11b, CD45, CD68, major histocompatibility complex (MHC) class I and MHC class II, but not for the astrocyte-specific markers, GFAP and glutamate aspartate transporter. SMK also showed phagocytic activity. In conclusion, this method resulted in a high-yield preparation of microglial cultures with ease and reproducibility.
authors.affiliation: Department of Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Seki
authors.forename: Satoshi O
authors.initials: SO
authors.lastname: Suzuki
authors.forename: Kenta
authors.initials: K
authors.lastname: Masui
authors.forename: Shiori
authors.initials: S
authors.lastname: Harada
authors.forename: Seiji
authors.initials: S
authors.lastname: Nakamura
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kanba
authors.forename: Toru
authors.initials: T
authors.lastname: Iwaki
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D002461:Cell Line, Transformed; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods / Q000592:standards; D017690:Cell Transplantation / Q000379:methods; D005260:Female; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D017628:Microglia / Q000166:cytology* / Q000637:transplantation; D008863:Microspheres; D010969:Plastics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
chemical_list: D000952:Antigens, Polyomavirus Transforming; D010969:Plastics; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins