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首页> 外文期刊>Cell Biology International Reports >A simple method for cell culture of a??Nemoa?? ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830)
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A simple method for cell culture of a??Nemoa?? ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830)

机译：一种简单的细胞培养方法？ ocellaris小丑鱼（Amphiprion ocellaris，Cuvier 1830）

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Worldwide, the a??Nemoa?? ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830) is one of the top three most exported ornamental fishes. It also served as a subject in various fields of study except for cell culture. This first report described a simple explant method for culturing cells from the vertebra of ocellaris clownfish. The fish was first anesthetised with iced cold water and decapitated. The body trunk was disinfected in isopropanol and washed in sterile PBS. The vertebra was aseptically excised, washed two times in PBS and minced in the dissection solution (PBS containing 250 IU/mL penicillin, 250a???μg/mL streptomycin, 50a???μg/mL gentamycin and 2.5a???μg/mL amphotericina??B). Then, the vertebral biopsies were washed three more times in PBS before being seeded in 25a??cm2 culture flasks containing 1.5a??mL of RPMIa??1640 supplemented with 20% FBS. A small amount of CO2 was injected into the flask before it was tightly capped and incubated at 28?°C in the regular incubator. When the monolayer reached 40a??50% confluence, the vertebral biopsies were dislodged together with the medium to initiate a new primary culture. The cell monolayer was subcultured with short, cold 0.05% trypsin. The Nemo cell line was grown in the medium containing 15% FBS. The cell line at passage 4 had the population doubling time of 39.6a??h and the cell line at passage 5 could be cryopreserved with 80% viability. This simple and reliable explant method has been applied successfully to culture cells of both marine and freshwater fishes for the prometaphase chromosome preparation.
机译：在全球范围内， ocellaris小丑鱼（Amphiprion ocellaris，Cuvier 1830）是出口量最大的三种观赏鱼之一。除细胞培养外，它还作为各个研究领域的主题。这份第一份报告描述了一种简单的外植体方法，用于从球藻小丑鱼的椎骨中培养细胞。首先将鱼用冰冷的水麻醉并斩首。躯干用异丙醇消毒，并用无菌PBS洗涤。无菌切除椎骨，在PBS中清洗两次，并在解剖溶液中切碎（含有250 IU / mL青霉素，250a-µg / mL链霉素，50a-µg / mL庆大霉素和2.5a-µg的PBS） / mL两性霉素？然后，将椎骨活检样品在PBS中洗涤3次以上，然后接种到25a -2 cm 2培养瓶中，该培养瓶中含有1.5a -3 mL RPMIa-1640并添加了20％FBS。在将烧瓶盖紧并在常规培养箱中于28°C孵育之前，将少量的CO2注入烧瓶中。当单层细胞达到40a→50％融合时，将椎骨活检与培养基一起移开，开始新的原代培养。用短的冷0.05％胰蛋白酶对细胞单层进行传代培养。 Nemo细胞系在含有15％FBS的培养基中生长。在第4代的细胞系的种群倍增时间为39.6aΔh，并且在第5代的细胞系可被冷冻保存，存活率为80％。这种简单可靠的外植体方法已成功应用于海水和淡水鱼类的细胞培养，用于前中期染色体的制备。

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