Dissociated Retinal Cell Culture
视网膜细胞的分离培养
发布: 2014年01月20日第4卷第2期 DOI: 10.21769/BioProtoc.1033 浏览次数: 15818
评审: Xuecai Ge
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参见作者原研究论文
The authors used this protocol in:
Nov 2012
Abstract
The retina is a relatively simple and accessible part of the central nervous system, making it a powerful model to study cell fate specification mechanisms. Multipotent retinal progenitor cells (RPCs) give rise to seven major classes of retinal cell types. Mechanisms regulating cell fate choice in the retina depend on both cell intrinsic and environmental factors, but their relative contribution to specific cell fate decisions remains unclear. Dissociated retinal cell cultures provide a great assay to study this problem. RPCs are cultured in serum-free and extract-free medium, providing the investigator with a control over the environment to address questions related to the effects of a particular molecule on the development of retinal neurons. In addition, dissociated cell cultures can be used to study the importance of cell intrinsic mechanisms by isolating RPCs from their normal environment (Cayouette et al., 2003; Jensen and Raff, 1997). The method described below is suitable for the clonal-density culture of RPCs. In such cultures, RPCs are isolated from each other and from the postmitotic neurons. They divide and differentiate into different retinal cell types to form small colonies, or “clones”. In a recent study, we found that these clones are indistinguishable from the clones that develop in situ in the retina, both in terms of cell number and cell type composition, suggesting that intracellular mechanisms play a key role in retinal development (Cayouette et al., 2003).
Keywords: Retina (视网膜)
Primary culture (原代培养)
Retinal neuron (视网膜神经元)
Retinal progenitor cells (视网膜祖细胞)
Clonal density (克隆密度)
Materials and Reagents
- Animals
We typically use retinas from albino Sprague Dawley rats. This method works for culturing rat retinas aged between embryonic day 17 to post-natal day 1. Younger and older retinal cells or retinal cells from mouse retinas do not seem to survive as well under these conditions, but can also be cultured. - DPBS (Life Technologies, catalog number: 14040 )
- Neurobasal medium (Life Technologies, catalog number: 21103-049 )
- DMEM/F12 medium (Life Technologies, catalog number: 10565-018 )
- B27 supplement (Life Technologies, catalog number: 17504-044 )
- Pen/Strep (P/S) (Life Technologies, catalog number: 15140-122 )
- Poly L-lysine (PLL) (Sigma-Aldrich, catalog number: P-4707 )
- Mouse Laminin (Life Technologies, catalog number: 23017-015 )
- Papain (Worthington Biochemical, catalog number: LS003126 )
- L-cystein crystal (Sigma-Aldrich, catalog number: C-7352 )
- Trypsin Inhibitor (Roche Diagnostics, catalog number: 109878 )
- 0.45 µm and 0.22 µm filters (Fisher Scientific, catalog number: SLHV033RS and SLGP033RS )
- Hoechst 33342 (Life Technologies, Molecular Probes®, catalog number : H3570 )
- “Incomplete” RGM medium
- Growth factors (see Recipes)
- 10x Lo Ovomucoid (see Recipes)
- 20x 4% BSA (Sigma-Aldrich, catalog number: A-4161 ) (see Recipes)
- 0.4% DNase (Worthington Biochemical, catalog number: LS002007 ) (see Recipes)
- Insulin (2.5 mg/ml) (Sigma-Aldrich, catalog number: I-6634 ) (see Recipes)
- cpt-cAMP (Sigma-Aldrich, catalog number: C-3912 ) (see Recipes)
- N-acetyl cysteine (NAC) (Sigma-Aldrich, catalog number: A-9165 ) (see Recipes)
- Forskolin (Sigma-Aldrich, catalog number: D-2438 ) (see Recipes)
- N2 Supplement (see Recipes)
- 4% Paraformaldehyde (Electron Microscopy Science, catalog number: 15710 ) (see Recipes)
- Apo-Transferrin (Sigma-Aldrich, catalog number: T-1147 ) (see Recipes)
- Progesterone (Sigma-Aldrich, catalog number: P-8783 ) (see Recipes)
- Putrescine (Sigma-Aldrich, catalog number: P-5780 ) (see Recipes)
- Sodium Selinite (Sigma-Aldrich, catalog number: S-5261 ) (see Recipes)
Equipment
- 35 mm petri dish (BD Biosciences, Falcon®, catalog number: 353001 )
- 50 ml conical tube
- CO2 incubator
- 15 ml conical tube
- 100 mm petri dish
- CO2 chamber
- Straight forcep
- Fine forcep (Fine Science Tools, catalog number: 11252-23 )
- Curved forcep (Fine Science Tools, catalog number: 91197-00 )
- Sharp scissor
- Laminar flow hood
- Water bath
- P1000 pipette
- Microscope
- Hemacytometer
- Centrifuge
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Jolicoeur, C. and Cayouette, M. (2014). Dissociated Retinal Cell Culture. Bio-protocol 4(2): e1033. DOI: 10.21769/BioProtoc.1033.
- Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W. Y., Harris, W. A. and Cayouette, M. (2012). Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina. J Neurosci 32(48): 17197-17210.
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分类
神经科学 > 发育 > 视网膜培养
细胞生物学 > 细胞分离和培养 > 细胞分离
细胞生物学 > 组织分析 > 组织分离
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