Retinal Explant Culture
视网膜外植体培养
发布: 2014年01月20日第4卷第2期 DOI: 10.21769/BioProtoc.1032 浏览次数: 21510
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
A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from embryonic animals and electroporated or infected with viral vectors, it is also a useful approach for the study of gene function at embryonic stages. Here, we present a retinal explant culture method that we have used extensively in various publications (Kechad et al., 2012; Cayouette et al., 2003; Cayouette and Raff, 2003; Elliott et al., 2008).
Keywords: Cell lineage (细胞谱系)
Neural progenitor (神经祖细胞)
Asymmetric division (不对称分裂)
Organotypic culture (器官培养)
Differentiation (区别)
Materials and Reagents
- Animals
We typically use retinas from albino Sprague Dawley rats or mice. Explants can be prepared from animals aged between embryonic day 12 to post-natal day 4. - DPBS (Life Technologies, catalog number: 14040 )
- DMEM (Life Technologies, catalog number: 10569-010 )
- Fetal bovine serum (FBS) (Wisent, catalog number: 080-450 )
- Penicillin/streptomycin (Life Technologies, catalog number: 15070 )
- 70% ethanol
- Fungizone antimycotic (Life Technologies, catalog number: 15290026 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- O.C.T. compound (Somagen, catalog number: 4583 )
- Explant medium
- 4% Paraformadehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 ) (see Recipes)
- 20% sucrose/OCT (1:1) (see Recipes)
- 20% sucrose solution (see Recipes)
Equipment
- Millicell organotypic insert (EMD Millipore, catalog number: PICMORG50 )
- 35 mm dish or 6 well plates
- 37 °C and 5% CO2 incubator
- 100 mm dish
- 150 mm dish
- Straight forcep
- FIne forcep (Fine Science Tools, catalog number: 11252-23 )
- Curved forcep (Fine Science Tools, catalog number: 91197-00 )
- Sharp scissor
- Sharp scapel (Fine Science Tools, catalog number: 10315-12 )
- Razor blade
- P1000 pipette
- CO2 chamber
- Cryomold 15 x 15 x 5 mm (VWR International, catalog number: CA60872-016 )
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). Retinal Explant Culture. Bio-protocol 4(2): e1032. DOI: 10.21769/BioProtoc.1032.
- Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W., 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
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