Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
胚胎大鼠海马和脑皮质神经元原代培养物的制备
发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2551 浏览次数: 17608
评审: Kae-Jiun ChangEmmanuelle BerretAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2017
Abstract
This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.
Keywords: Primary cultures (原代培养)
Hippocampal neurons (海马神经元)
Cerebrocortical neurons (脑皮质神经元)
Cell culture (细胞培养)
Background
The present protocol describes the preparation of primary cultures of rat hippocampal and cerebrocortical neurons, using Neurobasal medium supplemented with NeuroCultTM SM1 (Chen et al., 2008). The composition of NeuroCultTM SM1 is based on the formulation of the B27 supplement (Brewer et al., 1993), but the former cocktail was found to improve the quality of neuronal cultures, in part by replacement of apo-transferrin with holo-transferrin (Chen et al., 2008). Furthermore, the chemical composition of NeuroCultTM SM1 was described in more detail in the original publication, allowing a better control of the experimental conditions. Neuronal cultures prepared with chemically defined culture media are characterized by the presence of a low percentage of astrocytes. The proliferation of astrocytes in cultures maintained for longer periods of time, in order to allow differentiation of neurons, is prevented by adding the chemical inhibitor of mitosis 5-Fluoro-2’-deoxyuridine.
Materials and Reagents
- Petri dish, 55 mm Polysterene aseptic non-tissue culture treated (Labbox, catalog number: PDIP-06N-500 )
- Petri dish, 150 mm glass soda-lime (DWK Life Sciences, Duran, catalog number: 23 755 52 )
- 15 ml conical tube (Corning, Falcon®, catalog number: 352096 )
- 5 ml glass pipetes (VWR, catalog number: 612-4124 )
- 10 ml glass pipetes (VWR, catalog number: 612-4125 )
- 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
- Cell strainer 70 μm (Corning, Falcon®, catalog number: 352350 )
- Poly-D-lysine-coated multi-well plate (see step 16 in ‘Procedures’ for instructions)
- Coverslips #1.5 (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1014355110NR15 ; for 10 mm coverslips)
- Mixed cellulose ester filter, ME Range (ME 24), 0.2 μm pore size for filtration unit (GE Healthcare, Whatman, catalog number: 10406970 )
- Acetate cellulose filters of Ø25 mm, 0.20 μm, sterile (FRILABO, catalog number: 1520012 )
- Filtration unit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS0320-5033 )
- Stericup-GV, 0.20 µm, PVDF, 500/1,000 ml, radio-sterilized (Merck, catalog number: SCGVU10RE )
- Pregnant female Wistar rats (E17-E18 days gestation)
- Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
- Poly-D-lysine hydrobomide (Sigma-Aldrich, catalog number: P7886 )
- Boric acid (Merck, catalog number: 1001651000 )
- Nitric acid (Applichem, catalog number: 133255.1612 )
- Ethanol absolute 99.8+% (Fisher Scientific, catalog number: 10342652 )
- 5-Fluoro-2’-deoxyuridine (5-FDU) (Sigma-Aldrich, catalog number: F0503 )
- Potassium chloride (KCl) (AppliChem, catalog number: 131494.1211 )
- Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 1051041000 )
- Sodium chloride (NaCl) (Applichem, catalog number: 131659.1211 )
- Sodium bicarbonate (NaHCO3) (Acros Organics, catalog number: 123360010 )
- Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Merck, catalog number: 1065800500 )
- D(+)-Glucose monohydrate (VWR, catalog number: 24371.297 )
- Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
- HEPES (Fisher Scientific, catalog number: BP310-1 )
- Phenol red (Sigma-Aldrich, catalog number: P4758 )
- Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 27250018 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
- Minimum essential medium Eagle (MEM) (Sigma-Aldrich, catalog number: M0268 )
- Neurobasal Medium® (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
- NeuroCultTM SM1 Neuronal Supplement (STEMCELL Technologies, catalog number: 05711 )
- L-Glutamine (Sigma-Aldrich, catalog number: G8540 ; Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
- Glutamate (Sigma-Aldrich, catalog number: G1626 )
- Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
- Sodium hydroxide solution
- Hanks’ balanced salt solution (HBSS) (see Recipes)
- Trypsin solution (2 mg/ml) (see Recipes)
- 10% FBS (see Recipes)
- Neuronal Plating Medium (see Recipes)
- Supplemented neuronal culture medium (see Recipes)
- Boric acid solutions (see Recipes)
Equipment
- Large and small scissors
- Forceps with straight tip (Fine Science Tools, model: Dumont #5 )
- Forceps with curved tip (Fine Science Tools, model: Dumont #5 /45 forceps–Dumont standard tip)
- Pipetboy (Integra Biosciences, model: PIPETBOY acu 2 )
- Magnification glass
- Laminar flow hood
- Water-bath (Medingen Labortechnik, model: T100 )
- Phase contrast inverted microscope equipped with a 10x objective (Nikon Instruments, model: Eclipse TS100 )
- Hemocytometer (Marienfeld-Superior, catalog number: 0640010 )
- Humidified incubator
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Salazar, I. L., Mele, M., Caldeira, M., Costa, R. O., Correia, B., Frisari, S. and Duarte, C. B. (2017). Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons. Bio-protocol 7(18): e2551. DOI: 10.21769/BioProtoc.2551.
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分类
神经科学 > 细胞机理 > 细胞分离和培养
细胞生物学 > 细胞分离和培养 > 细胞生长
细胞生物学 > 细胞分离和培养 > 细胞分化
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