We graciously thank Dr. John Hayes for scripts; Drs. Eric Bradley and Christopher Del Negro for assistance with confocal microscopy; Drew Hughes, Laura Odorizzi, Alex Garafalo, Rebecca Lowden, and Liz MacMurray for their work in developing the project and providing preliminary data; Dr. Greg Smith for helpful suggestions on statistical analysis. This work was supported by an NIH grant (NINDS IR15N5067566-01) to MSS and a Howard Hughes Medical Institute Science Education Grant to the College of William and Mary.

All experiments are performed following protocols approved by the Institutional Animal Care and Use Committee at the College of William and Mary. Developmental stages referenced in this protocol are according to Nieuwkoop and Faber 46.
1. Dissection
<ol>
 <li>Allow the Cell Culture Medium and Calcium Magnesium Free Medium (CMF) to equilibrate to room temperature. You will also need 0.1X Marc's Modified Ringer's (MMR) pH 7.4-7.6.</li>
 <li>Sterilize the following items by applying ...

With its well-characterized cell types that are conserved across all vertebrates, the retina provides a useful model for studying the molecular-cellular processes governing cell type specification and differentiation. Primary cell culture affords a powerful method for investigating a wide range of processes including gene expression, protein dynamics, and calcium and electrical activity at the level of single cell resolution. Here we present a straightforward technique for primary cell culture from dissected presumptive ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Dissections and Culturing</td>
 </tr>
 <tr>
 <td>BD Falcon Easy Grip Tissue Culture Dishes, 35 mm</td>
 <td>Fisher</td>
 <td>08-772A</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable Polystyrene Petri Dishes, 60 x 15 mm</td>
 <td>Fisher</td>
 <td>0875713A</td>
 <td></td>
 </tr>
 <tr>
 <td>35 mm Nunclon Surface Petri Dishes (with Airvent)</td>
 <td>Fisher</td>
 <td>12-565-91</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont Fine Forceps (Dumostar #55)</td>
 <td>Fisher</td>
 <td>NC9341917 </td>
 <td></td>
 </tr>
 <tr>
 <td>Cellattice Micro-ruled plastic coverslip, 25 mm</td>
 <td>Fisher</td>
 <td>50-313-17 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl-m-Aminobenzoate Methanesulfonate Salt (MS-222)</td>
 <td>MP Biomedicals, LLC</td>
 <td>103106 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamicin Sulfate</td>
 <td>Enzo Life Sciences</td>
 <td>380-003-G025 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gibco Trypsin (1:250) Powder</td>
 <td>Life Technologies</td>
 <td>27250-018 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B from Clostridium histolyticum</td>
 <td>Roche</td>
 <td>11088831001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Nile Blue Sulfate (optional)</td>
 <td>Pfaltz &amp; Bauer</td>
 <td>N05550 </td>
 <td></td>
 </tr>
 <tr>
 <td>500 ml Vacuum Filter/Storage Bottle System, 0.22 &mu;m Pore 33.2 cm2 CN Membrane</td>
 <td>Corning</td>
 <td>430758</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Calcium Imaging</td>
 </tr>
 <tr>
 <td>Fluo-4 AM 1 mM Solution in DMSO, Cell Permanent</td>
 <td>Life Technologies</td>
 <td>F-14217</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-127 10% Solution in Water </td>
 <td>Life Technologies</td>
 <td>P-6866</td>
 <td></td>
 </tr>
 <tr>
 <td>LSM 510 Confocal Microscope System </td>
 <td>Zeiss</td>
 <td>Model Discontinued</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocking Reagent</td>
 <td>Roche</td>
 <td>11096176001</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Fluorescent in situ hybridization (FISH)</td>
 </tr>
 <tr>
 <td>Anti-Digoxigenin-POD, Fab Fragments</td>
 <td>Roche</td>
 <td>11207733910</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-DNP-HRP Antibody </td>
 <td>Perkin-Elmer</td>
 <td>NEL747A</td>
 <td></td>
 </tr>
 <tr>
 <td>Cy3 NHS ester</td>
 <td>GE Healthcare</td>
 <td>PA13101</td>
 <td></td>
 </tr>
 <tr>
 <td>NHS-Fluorescein </td>
 <td>Thermo Scientific</td>
 <td>46409</td>
 <td></td>
 </tr>
 <tr>
 <td>Formamide, Deionized</td>
 <td>Amresco</td>
 <td>0606-950ML</td>
 <td></td>
 </tr>
 <tr>
 <td>Torula RNA, Type IX</td>
 <td>Sigma-Aldrich</td>
 <td>R3629</td>
 <td></td>
 </tr>
 <tr>
 <td>Heparin Sodium Salt, from Porcine Intestinal Mucosa</td>
 <td>Sigma-Aldrich</td>
 <td>H3393-250KU</td>
 <td></td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>Sigma-Aldrich</td>
 <td>C3023</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 2. Specific reagents and equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td>Solution</td>
 <td>Reference</td>
 <td>Contents</td>
 </tr>
 <tr>
 <td>Cell Culture Medium</td>
 <td>Chang and Spitzer , 200954</td>
 <td>116 mM NaCl 
 0.67 mM KCl 
 2 mM CaCl2 
 1.31 mM MgSO4 
 4.6 mM Tris 
 1 % (v:v) Penicillin/Streptomycin 
 Adjust pH to 7.8 with HCl. 
 Filter sterilize by passing through a 0.22 &mu;m CN filter. 
 Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Calcium-Magnesium Free Medium (CMF)</td>
 <td>Gu et al., 199442; Gu and Spitzer, 199555</td>
 <td>116 mM NaCl 
 0.67 mM KCl 
 4.6 mM Tris 
 0.4 mM EDTA 
 1 % (v:v) Penicillin/Streptomycin 
 Adjust pH to 7.8 with HCl. 
 Filter sterilize by passing through a 0.22 &mu;m CN filter. 
 Store at 4 &deg;C. </td>
 </tr>
 <tr>
 <td>Maleic Acid Buffer (MAB)</td>
 <td>Sive et al., 200053 </td>
 <td>100 mM maleic acid 
 150 mM NaCl (pH 7.5).</td>
 </tr>
 <tr>
 <td>Marc's Modified Ringers (MMR), 10X</td>
 <td>Sive et al., 200053 </td>
 <td>100 mM NaCl 
 mM KCl 
 1 mM MgSO4 
 2 mM CaCl2 
 5 mM HEPES 
 pH adjusted to 7.4 with NaOH 
 0.1X MMR also contains 50 mg/ml gentamicin sulfate and pH is adjusted to 7.4 with NaOH.</td>
 </tr>
 <tr>
 <td>MEMFA Solution, 10X</td>
 <td>Sive et al., 200053</td>
 <td>0.1 M MOPS (Ph 7.4) 
 2 mM EGTA 
 1 mM MgSO4 
 3.7% formaldehyde 
 A 10X solution, without formaldehyde, can be stored at 4 &deg;C. Formaldehyde is added fresh (1/10 volume of a standard 37% stock). </td>
 </tr>
 <tr>
 <td>In situ Hybridization Buffer</td>
 <td>Sive et al., 200053</td>
 <td>50% Formamide 
 5X SSC 
 1 mg/ml Torula RNA 
 100 mg/ml Heparin 
 1X Denhart's Solution 
 0.1% Tween 20 
 0.1% CHAPS 
 10 mM EDTA.</td>
 </tr>
 </tbody>
</table>
Solutions. *Gentamicin, an antibiotic, is used in our MMR solutions while penicillin and streptomycin are used in our culture media.</td>
 </tr>
 </tbody>
</table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Examples of successfully dissected optic vesicles (stage 25) and retinae (stage 35) are shown in Figures 2E and 2J. While this protocol can be used at various stages of development, it is critical to obtain only retinal tissue to ensure accuracy for further experiments. Carefully remove the epidermis at all stages and ensure that your forceps do not puncture the retinal tissue. In stage 35 or older, the lens can be seen as a clear layer on top of the retina and can be removed by cautious scraping ...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

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13.7K ビュー.  College of William and Mary. アフリカツメガエルは、細胞運命の仕様および初代培養細胞内の個々の網膜細胞の生理機能を研究するための理想的なモデルシステムを提供します。ここでは、網膜組織を解剖し、カルシウム活性のために画像化し、in situハイブリダイゼーションにより分析される初代培養細胞を生成するための手法を提示する。