Xenopus laevis सेल भाग्य विनिर्देश और प्राथमिक सेल संस्कृति में व्यक्ति रेटिना की कोशिकाओं के शारीरिक कार्य के अध्ययन के लिए एक आदर्श मॉडल प्रणाली प्रदान करता है. यहाँ हम रेटिना ऊतकों विदारक और प्राथमिक सेल संस्कृतियों कि कैल्शियम गतिविधि के लिए imaged और स्वस्थानी संकरण द्वारा विश्लेषण पैदा करने के लिए एक तकनीक मौजूद है.
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 Müller 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).
इसकी अच्छी तरह से विशेषता कोशिका प्रकार है कि सभी रीढ़ भर में संरक्षित कर रहे हैं, रेटिना आणविक सेलुलर सेल प्रकार विनिर्देश और भेदभाव से संबंधित प्रक्रियाओं का अध्ययन करने के लिए एक उपयोगी मॉडल उपलब्?…
The authors have nothing to disclose.
हम कृपा से लिपियों के लिए धन्यवाद डॉ. जॉन Hayes, डीआरएस. एरिक ब्राडली और confocal माइक्रोस्कोपी के साथ सहायता के लिए क्रिस्टोफर डेल हबशी, आकर्षित किया ह्यूजेस, लौरा ODORIZZI, एलेक्स Garafalo, रेबेका Lowden, और लिज़ MacMurray परियोजना को विकसित करने और प्रारंभिक डेटा उपलब्ध कराने में अपने काम के लिए, सांख्यिकीय विश्लेषण पर उपयोगी सुझाव के लिए डा. ग्रेग स्मिथ. यह काम एक NIH एमएसएस के लिए (NINDS IR15N5067566 01) अनुदान और एक हॉवर्ड ह्यूजेस मेडिकल इंस्टीट्यूट विज्ञान शिक्षा विलियम और मरियम के कॉलेज के लिए अनुदान द्वारा समर्थित किया गया.
Name of the reagent | Company | Catalogue number | ||||||||||||||||||||||
For Dissections and Culturing | ||||||||||||||||||||||||
BD Falcon Easy Grip Tissue Culture Dishes, 35 mm | Fisher | 08-772A | ||||||||||||||||||||||
Disposable Polystyrene Petri Dishes, 60 x 15 mm | Fisher | 0875713A | ||||||||||||||||||||||
35 mm Nunclon Surface Petri Dishes (with Airvent) | Fisher | 12-565-91 | ||||||||||||||||||||||
Dumont Fine Forceps (Dumostar #55) | Fisher | NC9341917 | ||||||||||||||||||||||
Cellattice Micro-ruled plastic coverslip, 25 mm | Fisher | 50-313-17 | ||||||||||||||||||||||
Ethyl-m-Aminobenzoate Methanesulfonate Salt (MS-222) | MP Biomedicals, LLC | 103106 | ||||||||||||||||||||||
Gentamicin Sulfate | Enzo Life Sciences | 380-003-G025 | ||||||||||||||||||||||
Gibco Trypsin (1:250) Powder | Life Technologies | 27250-018 | ||||||||||||||||||||||
Collagenase B from Clostridium histolyticum | Roche | 11088831001 | ||||||||||||||||||||||
Penicillin-Streptomycin | Gibco | 15140-122 | ||||||||||||||||||||||
Nile Blue Sulfate (optional) | Pfaltz & Bauer | N05550 | ||||||||||||||||||||||
500 ml Vacuum Filter/Storage Bottle System, 0.22 μm Pore 33.2 cm2 CN Membrane | Corning | 430758 | ||||||||||||||||||||||
For Calcium Imaging | ||||||||||||||||||||||||
Fluo-4 AM 1 mM Solution in DMSO, Cell Permanent | Life Technologies | F-14217 | ||||||||||||||||||||||
Pluronic F-127 10% Solution in Water | Life Technologies | P-6866 | ||||||||||||||||||||||
LSM 510 Confocal Microscope System | Zeiss | Model Discontinued | ||||||||||||||||||||||
Blocking Reagent | Roche | 11096176001 | ||||||||||||||||||||||
For Fluorescent in situ hybridization (FISH) | ||||||||||||||||||||||||
Anti-Digoxigenin-POD, Fab Fragments | Roche | 11207733910 | ||||||||||||||||||||||
Anti-DNP-HRP Antibody | Perkin-Elmer | NEL747A | ||||||||||||||||||||||
Cy3 NHS ester | GE Healthcare | PA13101 | ||||||||||||||||||||||
NHS-Fluorescein | Thermo Scientific | 46409 | ||||||||||||||||||||||
Formamide, Deionized | Amresco | 0606-950ML | ||||||||||||||||||||||
Torula RNA, Type IX | Sigma-Aldrich | R3629 | ||||||||||||||||||||||
Heparin Sodium Salt, from Porcine Intestinal Mucosa | Sigma-Aldrich | H3393-250KU | ||||||||||||||||||||||
CHAPS | Sigma-Aldrich | C3023 | ||||||||||||||||||||||
Table 2. Specific reagents and equipment. |
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Solutions. *Gentamicin, an antibiotic, is used in our MMR solutions while penicillin and streptomycin are used in our culture media. |