Isolation, Purification, and Culture of Primary Murine Microglia Cells
原代小鼠小胶质细胞的分离、纯化和培养
发布: 2013年01月05日第3卷第1期 DOI: 10.21769/BioProtoc.314 浏览次数: 30102
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Abstract
The following is a detailed protocol for the isolation, purification and culture of murine brain microglia cells using neutral enzyme digestion and shaking. The protocol below is designed to isolate and culture a large number of purified inactivated microglia cells. Neutral enzyme digestion allows for minimal cellular damage and a higher cell recovery compared to other mechanical dissociative methods such as mincing. Cells cultured using this method will display the size and morphological features of microglia cells.
Keywords: Isolation (隔离)
Purification (净化)
Primary (初级的)
Microglia (小胶质细胞)
Materials and Reagents
- Five to eight fetal (1-3 days old) BALB/C mice (Harlan)
- Ice
- DMEM(Sigma-Aldrich, catalog number: D5796 )
- Fetal bovine serum heat inactivated (FBS) (Sigma-Aldrich, catalog number: F9665 )
- D-Hank’s balanced salt solution without phenol red (Life Technologies, InvitrogenTM, catalog number: H6648 )
- Trypsin / EDTA (0.05%/0.02%) (Life Technologies, InvitrogenTM, catalog number: 25300054 )
- Alcohol (70%)
- Poly-D-lysine solution (10 μg/ml) (Sigma-Aldrich, catalog number: L202 )
- 70% Alcohol
- Physiological saline (0.9%) (Sigma-Aldrich, catalog number: 07982-100TAB-F )
- Trypsin (0.25%) (Life Technologies, InvitrogenTM, catalog number: 25200-072 )
Two different concentrations of DMEM with FBS are used in order to maximize growth. The logic behind this is that the micrglia are more ‘sensitive’ after enzymic digestion. Therefore the use of DMEM with a higher concentration of FBS (i.e. 15%) provides additional growth factors and buffering agents after disaggregation and its use prior to enzymic expose is to allow the cells to acclimatize to this higher concentration and to optimize the cells under this improved growing condition. DMEM +FBS 10% is sufficient for culturing at all other periods.
Equipment
- Straight scissors
- Curved scissors
- Corneal scissors
- Straight forceps (small, x2)
- Curved artery forceps (x3)
- Beaker (100 ml, x2)
- Culture dish (30 mm, x2)
- Culture flask (25 cm2 and 75 cm2)
- Centrifuge Falcon tubes (15 ml and 50 ml)
- Serological pipettes (2 ml and 5 ml)
- Plastic transfer pipettes (3.5 ml)
- 70-μm nylon cell strainer
- Sterile gauze
- Incubator (37 °C; 5% CO2; humidified)
- Refrigerated centrifuge
- Magnifying glass and microscope
- UV light
- Culture flask with mixed glia cells
- Parafilm
- Alcohol burner
- Orbital shaker/Belly dancer (37 °C)
- 24-well tissue culture plate pre-coated with poly-D-lysine
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chen, X., Zhang, Y., Sadadcharam, G., Cui, W. and Wang, J. H. (2013). Isolation, Purification, and Culture of Primary Murine Microglia Cells. Bio-protocol 3(1): e314. DOI: 10.21769/BioProtoc.314.
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分类
免疫学 > 免疫细胞分离 > 胶质细胞
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