我们提出了一个有效的和易于使用的协议, 以制备斑马鱼胚胎的原细胞培养的转染和活细胞成像, 以及一个协议, 以准备初级细胞从成年斑马鱼的大脑。
斑马鱼的胚胎是透明的, 在母体外迅速发育, 因此可以在完整和发育的脊椎动物体内进行动态生物过程的良好成像。然而, 在整个坐骑中, 不同细胞类型和亚胞结构的形貌的详细成像是有限的。因此, 我们建立了一个有效和易于使用的协议, 培养活原细胞从斑马鱼胚胎和成人组织。
简言之, 2 的 dpf 斑马鱼胚胎 dechorionated、deyolked、消毒, 并与胶原酶分离成单细胞。在过滤步骤后, 主要细胞被镀到玻璃底部的菜肴和培育了几天。新鲜的文化, 尽可能多的长期 differenciated, 可用于高分辨率共焦成像研究。培养物中含有不同的细胞类型, 在聚 l-赖氨酸涂层中, 有横纹肌细胞和神经元突出。为了专门用荧光标记蛋白对亚细胞结构进行标记, 我们还建立了一种电穿孔协议, 允许将质粒 DNA 转染到不同类型, 包括神经元。因此, 在操作者定义的刺激, 复杂细胞行为, 和细胞内动力学的主要斑马鱼细胞可以评估与高空间和时间分辨率。此外, 利用成年斑马鱼的大脑, 我们证明了所描述的离解技术, 以及基本的培养条件, 也为成年斑马鱼组织工作。
斑马鱼 (斑马斑马,斑马) 是一个流行的模型脊椎动物为许多领域的基础和生物医学研究1。斑马鱼胚胎发育迅速,前子宫, 是透明的, 并适合显微镜下, 从而提供了良好的先决条件, 研究脊椎动物的发展, 在一个活生生的有机体。由于斑马鱼2的遗传驯良, 许多稳定的转基因报告线与细胞类型特异表达的各种荧光标记已建立允许观察特定的细胞群体。斑马鱼社区提供了各种各样的所谓的 Gal4-driver 线, 携带转基因表达合成 Kal4TA4 (或 KalTA3-equivalent GalFF) 基因与 Gal4-DNA-binding 领域的酵母融合到病毒转录激活域在细胞类型特异增强剂的控制下。这些驱动程序线被交叉到执行器线, 其中运载转基因由定义的上游激活序列 (无人驾驶) 融合到一个记者基因。Kal4TA4 蛋白结合到无人研究的元素, 从而激活细胞类型选择表达的报告基因3,4。这种方法允许对双转基因动物中几乎所有可用增强因子和报告元素进行高度多样的组合研究。
然而, 在一个整体和不断变化的胚胎中, 聚焦于单个细胞或其亚型含量的深度实时成像是有限的。为了解决特定细胞生物学问题的最高分辨率, 使用细胞培养往往是可取的。斑马鱼的一些细胞线存在, 但它们被认为是重选择5,6,7 , 他们的繁殖往往是耗时的。此外, 所有可用的细胞系都是成纤维细胞, 限制实验使用细胞培养到一种类型的单元。因此, 我们建立了一个有效和易于使用的协议, 直接从斑马鱼胚胎和斑马鱼的大脑中制备初级细胞, 以及提高文化的长寿和扩大栽培的多样性的方法。单元格类型。此外, 我们提出了一个程序, 染胚胎原细胞与表达结构的荧光细胞器标记。因此, 细胞形态和亚细胞结构可以用高的空间和时间分辨率来进行分析, 从而保持其关键特征。
在这里, 我们提出了两种不同的协议, 培养主要细胞从 2 dpf 斑马鱼胚胎或成年斑马鱼的大脑。
从 2 dpf 斑马鱼中制备原代细胞培养物, 对于任何有基本细胞培养技术经验的人来说, 都是相对容易执行的。然而, 为了获得良好和可重现的结果, 足够数量的胚胎作为起始材料是至关重要的 (100 是最低的)。在胚胎的饲养过程中, 必须避免所有可能的污染源, 以尽可能降低细菌和寄生虫的?…
The authors have nothing to disclose.
我们感谢 t. Fritsch, a. Asseburg, i. 林德和 S. m。Tokarski 为优秀的动物护理和技术支持。我们感谢 Köster 实验室的所有成员进行了激烈而有益的讨论。我们感激地承认德意志 Forschungsgemeinschaft (高 1949/5-1) 和联邦下萨克森, Niedersächsisches Vorab (VWZN2889) 提供的资金。
Fish lines | ||||||
AB (wild-type) | established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC) | |||||
Tg(ptf1a:eGFP)jh1 | stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007) | |||||
Tg(XITubb:DsRed)zf148 | stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed (Peri and Nüsslein-Volhard, 2008) | |||||
Name | Company | Catalog Number | Comments | |||
Equipment | ||||||
centrifuge | Eppendorf | model 5804 R | ||||
ChemiDoc MP imaging system | BioRad | model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos | ||||
confocal laser scanning microscope | Leica microsystems | model SP8, equipped with 28 °C temperature box and a 63 x objective | ||||
epifluorescent microscope | Leica microsystems | model DM5500B, equipped with 28 °C temperature box and a 40 x objective | ||||
Gene Pulser Xcell with capacitance extender | BioRad | 1652661 | electroporation device | |||
Horizontal shaker | GFL | model 3011 | ||||
incubator for cell culture (28 °C) | Memmert | model incubator I | ||||
incubator for embryos (28 °C) | Heraeus | type B6120 | ||||
light microscope | Zeiss | model TELAVAL 31 | ||||
micro pipettes | Gilson | |||||
sterile work bench | Bio Base | with laminar flow and UV light | ||||
tweezers | Dumont | Style 5, Inox | ||||
vertical tube rotator | Labinco B.V. | model LD-79 | ||||
Name | Company | Catalog Number | Comments | |||
Software | ||||||
Image Lab Software | BioRad | for the ChemiDoc MP imaging system from BioRad | ||||
ImageJ | National Institutes of Health | used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016). | ||||
LAS X | Leica Microsystems | for both confocal and epifluorescent microscopes from Leica Microsystems | ||||
Name | Company | Catalog Number | Comments | |||
Plasmids | ||||||
pCS-DCX-tdTomato | Köster Lab | # 1599 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-eGFP | Köster Lab | # 7 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-H2B-mseCFP | Köster Lab | # 2379 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-mClover | Köster Lab | # 3865 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-MitoTag-YFP | Köster Lab | # 2199 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-ss-RFP-KDEL | Köster Lab | # 4330 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pCS-VAMP1-mCitrine | Köster Lab | # 2291 | based on the backbone pCS2+ (Rupp et al., 1994) | |||
pSK-UAS:mCherry | Köster Lab | # 1062 | based on the pBluescript-backbone of Stratagene | |||
Plasmid numbers refer to the database entries of the Köster lab. Plasmids are available upon request. | ||||||
Name | Company | Catalog Number | Comments | |||
Plastic and glass ware | ||||||
BD Falcon Cell Strainer (40 µm) | FALCON | REF 352340 | distributed by BD Bioscience, used as “landing net” to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium | |||
1.5 mL reaction tubes | Sarstedt | 72690550 | ||||
24-well plate | Sarstedt | 83.3922 | ||||
50 mL falconic tube | Sarstedt | 62.547.004 | ||||
96-well plate | Sarstedt | 83.3924.005 | ||||
EasyStrainer (40 µm) | Greiner Bio-One | 542 040 | with venting slots; used to filter cells after collagenase-mediated dissociation | |||
electroporation cuvette (0.4 cm) | Kisker | 4905022 | ||||
glass coverslips | Heinz Herenz Medizinalbedarf GmbH | 1051201 | ||||
Microscope slides | Thermo Fisher Scientific (Menzel Gläser) | 631-0845 | ||||
Neubauer chamber | Henneberg-Sander GmbH | 9020-01 | ||||
Pasteur pipettes (plastic; 3 mL) | A. Hartenstein | PP05 | ||||
Petri dishes (plastic; diameter 10 cm) | Sarstedt | 821473 | for zebrafish embryos | |||
pipette tips | Sarstedt | Blue (1000 µl): 70762; Yellow (200 µl): 70760002; White (10 µL): 701116 | ||||
sterile cell culture dishes (plastic; diameter 3 cm) | TPP Techno Plastic Products AG | 93040 | ||||
sterile cell culture dishes (plastic; diameter 6 cm) | Sarstedt | 72690550 | ||||
sterile Petri dishes (plastic; diameter 10 cm) | Sarstedt | 83.3902 | for brain dissection | |||
Name | Company | Catalog Number | Comments | |||
Chemicals and Reagents | ||||||
sodium chloride | Roth | 0601.1 | ||||
4 % paraformaldehyde in 1 x PBS | Sigma-Aldrich | 16005 | ||||
4',6-diamidino-2-phenylindole (DAPI) | Thermo Fisher Scientific | D1306 | ||||
calcium nitrate tetrahydrate | Sigma-Aldrich | C1396 | ||||
ethanol p.a. 100% | Sigma-Aldrich | 46139 | ||||
goat α-mouse IgG (Fc specific) FITC conjugated | Thermo Fisher Scientific | 31547 | ||||
HEPES | Roth | 9105.4 | ||||
high vacuum grease | DOW CORNING | 3826-50 | silicon grease used for self-made glass bottom dishes | |||
magnesium sulfate heptahydrate | Merck | 105886 | ||||
methylene blue | Serva | 29198.01 | ||||
Monoclonal Anti-Tubulin, Acetylated antibody | Sigma-Aldrich | T6793 | ||||
Aqua-Poly/Mount (mounting medium) | Polyscience | 18606 | ||||
poly-L-lysine | Biochrom | L 7240 | ||||
potasssion chloride | Merck | 104938 | ||||
Skim milk | Roth | 68514-61-4 | ||||
Texas Red-X Phalloidin | Thermo Fisher Scientific | T7471 | ||||
Tricaine | Sigma-Aldrich | E10521 | Synonym: Ethyl 3-aminobenzoate methanesulfonate | |||
Triton X-100 | BioRad | 1610407 | ||||
Trypan Blue | Gibco by Life Technologies | 15250061 | ||||
Name | Company | Catalog Number | Comments | |||
Enzymes | ||||||
collagenase (Type 2) | Thermo Fisher Scientific | 17101015 | dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 °C | |||
pronase (from Streptomyces griseus) | Roche | 11459643001 | distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 °C | |||
Name | Company | Catalog Number | Comments | |||
Medium and solutions for cell culture | ||||||
1 x PBS (Dulbecco's Phosphate Buffered Saline) | Gibco by Life Technologies | 14190-169 | distributed by Thermo Fisher Scientific | |||
CO2-independent medium | Gibco by Life Technologies | 18045054 | distributed by Thermo Fisher Scientific | |||
filtrated bovine serum (FBS) | PAN-Biotech | individual batch | ||||
glutamine 100 x | Gibco by Life Technologies | 25030081 | distributed by Thermo Fisher Scientific | |||
Leibovitz's L-15 medium | Gibco by Life Technologies | 11415049 | distributed by Thermo Fisher Scientific | |||
PenStrep (10,000 units/mL) | Gibco by Life Technologies | 15140148 | distributed by Thermo Fisher Scientific |