原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜
Summary
This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.
Abstract
原代大鼠新生心肌细胞是有用的基本体外心血管研究，因为它们可以很容易地分离出大量的单一步骤。由于在显微镜技术的进步是相对容易捕捉到活细胞的图像进行调查的实时有关的光毒性的细胞的细胞事件以最小的关注的目的。本协议描述了如何利用共聚焦旋转盘显微镜下慢病毒和腺病毒转导调节细胞的特性采取原代大鼠乳鼠心肌细胞的活细胞慢速拍摄的图像。两种不同类型的病毒的应用可以更容易地实现合适的转导率和表达水平的两个不同的基因。以及聚焦活细胞图像可以使用显微镜的自动对焦系统，可以保持稳定的聚焦长的时间段来获得。应用这种方法，外源性工程师的功能表达在培养的原代细胞编蛋白进行分析。此外，该系统可用于通过化学式调制器的使用的siRNA以及检查基因的功能。
Introduction
原代大鼠新生心肌早已被用于体外 1调查心肌功能。他们很容易从幼鼠由几个公认的方法2-4隔离开来。最常见的方法使用胶原酶或胰蛋白酶消化之前，细胞分离心脏的结缔组织。研究人员还开发了从成年啮齿动物5-8以及新生小鼠9,10分离心肌细胞的方法。
这个协议描述了从新生大鼠幼仔分离心肌细胞，采用两步酶消化过程的方法。胰蛋白酶是第一次使用O / N在4℃，然后纯化胶原酶在37°C心脏组织用胰蛋白酶O / N的潜伏期在4℃可减少相比，使用顺序孵化在一个温暖的酶液2种方法需要收获细胞的步骤。此外，通过使用纯化的共llagenase而不是粗酶液，批与批之间的变化可以被消除，从而提供增强的可重复性。
特定蛋白质的功能性研究经常采用利用腺病毒11-13和/或14-16的慢病毒的蛋白质表达系统。 [忠告]病毒的生产和操作应根据美国国立卫生研究院的指引进行。
腺病毒不整合到宿主基因组中。它具有转导在大多数细胞类型，包括分裂细胞和非分裂细胞，以及原代细胞和建立的细胞系中非常高的效率。这使得腺病毒的可靠载体用于基因表达。高水平的腺病毒载体编码的蛋白的48小时内下列传导开发，并且它们可以持续数周17。然而，一个缺点是使用的腺病毒蛋白质的表达是一种重组腺的发展novirus是既复杂又费时。这个缺点在部分解释了为什么许多研究人员已经转向慢病毒重组基因的表达。与腺病毒的构建，产生慢病毒结构非常简单快捷。虽然慢病毒通常具有转导的低效率比腺病毒，在两个分裂和非分裂细胞，它们整合到宿主基因组中。因此，转基因的表达对于慢病毒比为腺病毒更稳定。
由于在显微镜领域的技术进步，这是很容易捕捉到细胞中表达重组蛋白的活细胞的图像。这甚至在收购视频速率的速度也是如此。这使得研究者确定如何具体改变在感兴趣的蛋白质在功能上影响实时细胞。共聚焦旋转盘显微镜有，使它成为LIV最佳技术的几个关键特性Ë细胞成像18,19。横河电机旋转的光盘允许更快速图像获取，而在同一时间利用少得多的激光功率比点扫描共聚焦显微镜。这两个特殊的特性是由于旋转的盘，其中包含通过该激光同时传递到许多样品的共焦孔。在采集过程中，磁盘本身旋转快速，持续18-20。通过使用显微镜的自动对焦系统，稳定的焦点被维持在很长一段时间。这使研究人员能够采取有重点的活细胞图像。采集的图像回放的电影文件。图像是使用图像分析软件，如ImageJ的21,22，斐济23，或其它市售软件进行分析。
Protocol
1，隔离乳鼠心肌细胞用Dulbecco改进的Eagle培养基（DMEM）和补充有胎牛血清（FBS）和青霉素/链霉素（P / S）作为培养基的最低必需培养基（MEM）。添加溴脱氧尿苷（BrdU）标记为介质，以防止成纤维细胞生长为隔离后的第2天。 基础培养基 FBS 的BrdU（MM） P / S（U / ml的） 选择 DMEM 10?…
Representative Results
为了说明的技术中，慢病毒编码EGFP（增强型绿色荧光蛋白）标记的Cx43（连接蛋白43）或突变的Cx43蛋白32，来表达EGFP-标记的蛋白质在细胞和腺病毒编码FGFR1DN（成纤维细胞生长因子受体1显性失活）被用来关闭FGF信号在细胞33-35。三天之后的心肌细胞的分离，该分离的心肌细胞转导的慢病毒，以表达EGFP-标记的蛋白质在细胞内。然后后的慢病毒转导后3天，分离的心肌细胞进一步转导?…
Discussion
从新生大鼠原代心肌早就被用来研究心肌细胞功能的体外 。这个协议描述了用于从幼鼠采用两步酶消化法新生心肌细胞的分离方法，首先用胰蛋白酶O / N消化，在4℃，然后纯化的胶原酶。采用纯化的胶原酶步骤的一个优点在于，同一等级的酶被用于所有隔离。因此，分离的细胞的质量和数量是从实验的实验一致。
给定与腺病毒相关的，如果细胞培养转导的高效率是必?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Alengo Nyamay’antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.
Materials
| 1 scissors for decapitation | WPI | 501749 | Autoclave before use |
| 1 fine scissors for heart isolation and chopping | WPI | 14393 | Autoclave before use |
| 2 fine forceps (Dumont No. 5) | Sigma | F6521 | Autoclave before use |
| Three sterilized 10 cm plastic dishes | Sigma | CLS430165 | for hearts isolation |
| 3.5 cm glass bottom culture dishes | MatTek | P35G-1.5-20-C | for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative. |
| 3.5 cm glass bottom culture dishes | MatTek | P35G-0.17-14-C | for TIRF or high resolution image |
| Ethanol solution, 70 % (v/v) in water | Sigma | E7148 | |
| 2% gelatin | Sigma | G1393 | |
| One sterilized 10 cm plastic dish | Sigma | CLS430165 | for trypsinization |
| Aluminium foil | any brand | ||
| parafilm | Sigma | P7543 | |
| Two 10 cm plastic cell culture dish | Sigma | CLS430165 | for selection |
| Auto pipette | Drummond Scientific | 4-000-300 | for trituration |
| Cell counter | |||
| Cellometer, automated cell counter | nexcelom | to check and count cells | |
| Microscope and Hematocytometer | any brand | to check and count cells | |
| Trypan Blue Solution, 0.4% | invitrogen | 15250-061 | |
| CO2 incubator | Sanyo | MCO-19AIC | |
| incubating orbital shaker | Sigma | Z673129 | to shake heart tissue with collagenase at 37 C at 170-200 rpm |
| 10 mg/ml BrdU solution | BD Pharmingen | 550891 | |
| DMEM, High Glucose | invitrogen | 41965039 | Mix medium as indicated in the protocol and warm before use |
| MEM | invitrogen | 31095029 | Mix medium as indicated in the protocol and warm before use |
| Fetal Bovine Serum | invitrogen | 26140079 | |
| Penicillin-Streptomycin (10,000 U/mL) | invitrogen | 15140-122 | |
| Section 2 lentiviral transduction | |||
| three packaging plasmids | |||
| pMDLg/pRRE | Addgene | 12251 | |
| pRSV-Rev | Addgene | 12253 | |
| pMD2.G | Addgene | 12259 | |
| The lentiviral transfer vector, pLVX-IRES-Puro | Clontech | 632183 | |
| Opti-MEM (serum-free medium) | invitrogen | 31985070 | |
| transfection reagent | |||
| polyethyleneimine“Max”, (Mw 40,000) – High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form) | Polysciences | 24765-2 | It can be substituted with X-tremeGENE 9 from Roche |
| X-tremeGENE 9 | Roche | 6365779001 | substitute for PEI as transfection reagent |
| chloroquine | Sigma | C6628 | dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 μM Chloroquine. |
| HEPES | Sigma | H3375 | |
| 10 ml Luer-Lok syringe, sterilized | BD | 309604 | |
| 0.45 um filters | Sigma | F8677 | use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus. |
| Hexadimethrine bromide | Sigma | H9268 | dissolve in water and make 8mg/ml stock solution, then filter it to sterilize. |
| polyethylene glicol 6000 | Sigma | 81260 | |
| Sodium chloride | Sigma | S9888 | |
| sodium hydroxide | Sigma | S5881 | |
| Section 3 Adenoviral transduction | |||
| HEK 293T cells | ATCC | CRL-11268 | |
| Some 10 cm cell culture dishes | Sigma | CLS430165 | |
| 96-Well Microplate with lid, flat-bottom, tissue culture, sterile | BD Falcon | 353072 | for titration |
| Multichannel piptte, 10-100 ul, 8-channel | eppendorf | 3122 000.035 | |
| Section 4 live cell imaging | |||
| Spinning disk confocal microspopy | PerkinElmer | L7267000 | |
| a temperature controlled chamber | any brand | to keep temperature at 37 C | |
| a CO2 environmental system | any brand | optional to maintain CO2 concentration optimal | |
| Medium | |||
| CO2 Independent Medium, No Glutamine | invitrogen | 18045-054 | for long time time-lapse imaging |
| DMEM, High Glucose, HEPES, no Phenol Red | invitrogen | 21063-029 | for long time time-lapse imaging |
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Cite This Article
Sakurai, T., Lanahan, A., Woolls, M. J., Li, N., Tirziu, D., Murakami, M. Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy. J. Vis. Exp. (88), e51666, doi:10.3791/51666 (2014).