隔间文化背根神经节神经元的制备及维修 (Translated to Chinese)

Cite this Article: 隔间文化背根神经节神经元的制备及维修

F. Pazyra-Murphy, M., A. Segal, R. Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures. J. Vis. Exp. (20), e951, doi:10.3791/951 (2008).

Abstract: 隔间文化背根神经节神经元的制备及维修

神经元的延伸，从细胞体拆下来支配靶组织，靶源性生长因子神经元的存活和功能的要求的轴突过程。神经营养因子具体要求，以维持生存和支配的感觉神经元的分化，但如何将这些目标源性神经营养因子的支配神经元细胞体沟通的问题已经超过30年的一个活跃的研究领域。神经营养因子的信号如何到达细胞体的最普遍接受的模型的建议，信令内涵体进行这个信号沿着轴突逆行。为了研究逆行运输，文化系统的最初设计由罗伯特Campenot，其中胞体从它们的轴突隔离。技术准备培养的感觉神经元概括选择性刺激神经元的终端发生以下靶源性神经营养因子在体内释放这些隔间商会。逆行信号的事件，需要长距离微管依赖逆行运输的神经退行性疾病的治疗具有重要意义。

Protocol: 隔间文化背根神经节神经元的制备及维修

试剂的制备

胶原涂层 ：胶原蛋白外衣P35组织培养板，并放置在烤箱在37 ° C时2天前润滑的分隔。最后胶原蛋白的浓度应在0.71毫克/毫升0.001 ñ盐酸稀释。然后，添加1毫升每盘的混合物。
油脂装载机 ：为了填补油脂装载机，一个60ml的注射器必须先与康宁真空润滑脂填充。使用注射器填补油脂装载机，包装箔，然后高压灭菌45分钟。
聚四氟乙烯分隔的分频器，可每次实验后重新使用，但首先必须正确地清洗。从板卸下分配器，擦去所有剩余的油脂和硫酸为2天。从酸中取出后，冲洗3倍的水，煮沸20分钟，晾干，在一个玻璃P100培养皿和20分钟高压灭菌的地方。
N2 -甲基纤维素 ：称取甲基纤维素1.5克，放置在500毫升的瓶子。添加搅拌棒，高压灭菌20分钟就干（这一点所有的工作必须是无菌的）。接下来，添加无血清媒体（N _2）500毫升，在寒冷的房间中，搅拌直至溶解。分装成50毫升conicals和冻结在-20 ° C。对于股票的工作，分装50毫升conicals的一成1ML管和冻结在-20 ° C。
DRG的媒体 ：DMEM培养液，5％热灭活马血清，1％青霉素链霉素。
100ng/mL DRGN媒体的神经生长因子（NGF）和脑源性神经营养因子（BDNF）的股票浓度为1mg/ml。稀释成每个人的背根神经节媒体神经营养因子1:10,000。文化可以仅在NGF的增长，这改变了神经元在文化生存的补充。

注意：当需要时，浓度（阿糖胞苷）ARAC 0.3uM最终浓度在1微米和使用。这将抑制雪旺氏细胞和其他神经胶质细胞的生长。
在10ng/ml DRGN媒体 ：100ng/mL DRGN媒体（1:10）稀释与DRG的媒体。

设置所需的工具图1 。

隔间商会成立（启动前清扫这个过程1-2天）

请在涂胶原蛋白P35盘向外运动中的划伤。
N2 -甲基广场30ul从头中间。设置菜放在一旁，直到分频器是润滑。
将一个23号鲁尔存根适配器油脂装载机。抓住一双90 °角止血特氟隆分压器和奠定它在显微镜下所面临的分压器持平。分压器跟踪与油脂，确保每次适配器适配器是从上一步中的油脂插入一个新的起点上，以便有连续线的油脂（见图表）。一旦油脂被应用到整个分，依次准备P35菜肴之一倒挂，并把它使N2 -甲基中间的车厢。按底部的菜用的镊子。请务必按分压器内部四角（左上角，右下角，右上角，左下角，在图所示的“X”）。

图2：润滑分配器的步骤

注：重要的是用力按压足够的油脂，使一个完全密封的菜，但如果添加太多的压力，轴突不会跨到侧面车厢。

拿起止血，翻过来，松开分压器。最后，坚决附加分压器中间车厢底部聚焦显微镜下的菜。随着油脂加载器，使一个小的障碍（0.25厘米），因此，一旦细胞被放置在中间舱，他们不能泄露出去。
后成立了文化，在每个侧面车厢DRG的媒体和地点在将保持在该细胞的孵化器。让文化坐了几个小时，然后检查有无泄漏。如果媒体已泄露到中间的车厢，然后文化是不可用的。

注：当第一次学习这种技术，它是重要的，成立超过一个实验需要的文化，几个将漏水。

图3：“好VS漏”文化

在隔间文化保持背根神经节神经元

第1天 ：100ng/mL DRGN媒体+ ARAC一边车厢，更换DRG的媒体。进行清扫，并添加细胞中心室（100000细胞）。
第2天 ：添加在10ng/ml媒体+ ARAC聚四氟乙烯分频器外，直到媒体中心舱的油脂阻隔与交流流体流动。
第3天 ：替换在一旁车厢媒体100ng/mL DRGN省略在10ng/ml DRGN省略ARAC ARAC和环绕声。
第6天 ：1ng/mL + ARAC和DRG的媒体+ ARAC环绕在一旁车厢替换的媒体。
第九天 ：进行试验使用。

图4：胞体和远端轴突的免疫组化图像

注意：改变媒体时，重要的是从每一方舱顶部的吸液。此外，从中间的车厢本身永远不会改变媒体，只有从环绕，并让它流在进入中心的油脂阻隔。

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Discussion: 隔间文化背根神经节神经元的制备及维修

在这段视频中，我们已经演示了如何编写和维护用于培养DRG神经元的隔间商会。处理得当，这个系统可以从轴突细胞体内分离，以研究神经营养因子，其中跨长轴突信号机制。由于车厢之间的隔离流体，它允许没有受到影响的其他车厢选择性刺激或一室的治疗。隔间腔文化可以支持其他类型的细胞，包括从颈上神经节，视网膜神经节神经元和皮层神经元交感神经元。空间理解神经营养因子信号转导的神经退行性疾病的治疗提供了新的见解。一些神经退行性疾病，包括老年痴呆症，亨廷顿氏症和运动神经元疾病，都与轴突运输缺陷。最近的研究中使用了微流体的商会，而不是这些隔间商会。 ^4,5微流体商会成像分析的几个优点。

此前的研究测试，这些文化的能力，以防止扩散之间的轴突和胞体车厢^1,3,6。这可以很容易地通过添加低浓度的，如台盼蓝染色仅一室，并寻找染料的扩散测试。应在24小时内很少或根本没有扩散可见。

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Disclosures: 隔间文化背根神经节神经元的制备及维修

Acknowledgements: 隔间文化背根神经节神经元的制备及维修

我们想感谢卡塔琳娜Cosker和斯蒂芬妮Courchesne有益的讨论。

Materials: 隔间文化背根神经节神经元的制备及维修

Name	Type	Company	Catalog Number	Comments
collagen	Reagent	BD Biosciences	354249
N2-methylcellulose 400CPS	Reagent	Sel-Win Chemicals
Teflon divider	Other	Tyler Research	CAMP10	many other types of dividers are available
Pin rake	Tool	Tyler Research	Camp-PR
Grease loader	Tool	Tyler Research	Camp-GLSS
DMEM	Reagent	Fisher Scientific	MT10017CV
NGF	Reagent	PeproTech Inc	450-01
BDNF	Reagent	PeproTech Inc	450-02
High vacuum grease	Reagent	Fisher Scientific	14-635-5D
AraC	Reagent	Sigma-Aldrich	C-1768
23 gauge luer stub adapter	Tool	Fisher Scientific	427565
90° angle hemostats	Tool	Roboz Surgical Instruments Co.	RS-7035

References: 隔间文化背根神经节神经元的制备及维修

1. Campenot, RB. Independent Control of the Local Environment of Somas and Neurites. Methods in Enzymology 58 302-7, (1979).

2. Watson, FL., et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience 4 981-88, (2001).

3. Heerssen, HM., et al. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nature Neuroscience 7 596-603, (2004).

4. Taylor, AM., et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods 2 599-605, (2005).

5. Park JW., et al. Microfluidic culture platform for neuroscience research. Nat Protoc. 4 2128-36, (2006).

6. Ure DR., et al. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neuroscience 4 1282-90, (1997)

Ask the Author: 隔间文化背根神经节神经元的制备及维修

18 Comments

I have just watch your presentaion on the Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures and it was very impressive. I am intersted in the function of the methylcellulose, does it provide a space through which the axons can pass, do the cell bodies settle on the collagen surface.

Regards

Paul

Posted by: Paul MillnsNovember 19, 2008, 7:27 AM

Hi Paul-
Thank you for your comment. The cell bodies do settle onto the collagen surface. As for the small amount of N2-methylcellulose that is placed on the center of the scratch, the media acts as a space for the axons to extend easily on and the methylcellulose is used to thicken the media slightly.

Good Luck,
Maria

1.1

Posted by: AnonymousDecember 15, 2008, 3:06 PM

Hi,

your method looks simple and powerfull.

It's possible to make an immuno staining without to remove the teflon divider? Does the divider cause troubles during immunofluorescence staining for example?

Regards

Giuseppe

Posted by: AnonymousDecember 13, 2008, 7:35 AM

Hi Giuseppe-
Thank you for your comment. Fix, wash and add antibodies as usual within the compartments for immunostaining. Remove the divider, carefully wipe away excess grease and coverslip before imaging.

Good Luck,
Maria

2.1

Posted by: AnonymousDecember 15, 2008, 3:52 PM

Hi Maria,

I had a question about immunostaining. So in reference to your above comment, you keep the teflon dividers in place when you fix and add antibodies, and only remove it at the end of immunostaining? Is anything else different?

Thanks,
Supraja.

2.1.1

Posted by: SuprajaOctober 22, 2010, 7:01 PM

Hi Supraja-
Yes, only remove the divider at the end of the immunostaining. Everything else is done exactly the same.

Good Luck,
Maria

2.1.1.1

Posted by: AnonymousOctober 25, 2010, 3:42 PM

Hi,

beautiful work, congratulations!

Have you tried to cultivate adult DRG also?

Best regards and good luck!

Otilia

Posted by: Otilia ObrejaFebruary 5, 2009, 6:34 AM

Hi Otilia-

Thank you for your comment. We have not tried cultivating adult DRGs in this system.

Good Luck,

Maria

4.1

Posted by: AnonymousMarch 19, 2009, 12:03 PM

Dear Otilia,

I too was really impressed by Maria's work and wanted to use the technique for my work. I just wanted to let you know that I have been using adult DRGs in this system and it still works brilliantly.

Best regards,

Philippa

4.2

Posted by: AnonymousSeptember 2, 2009, 1:02 PM

Thanks a lot, Philippa!

4.2.1

Posted by: OtiliaJanuary 18, 2010, 4:08 PM

Hi,

I hvae not been able to watch the video. Could you please email me a copy.

Sincerely,

Supinder Bedi, Ph.D.

University of Texas, Houston

Posted by: Supinder BEdiFebruary 11, 2009, 3:39 PM

Hi,

We are very interested in your method, that’s great!

We have one question: on day1, how do you "perform dissection and add cells to center compartment (100,000cells)"?

Can you please provide more details?

Best regards

Posted by: Yi DingMarch 5, 2009, 3:52 PM

Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use?

The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen doesn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using.

Any suggestions would be really appreciated.

Best regards, Philippa

Posted by: Philippa M.March 19, 2009, 7:25 AM

Hi Philippa-

Thank you for your comment. We only use polystyrene cell culture dishes that have been coated with collagen. It is impossible to make the necessary scratches on the glass. We often do immunostaining and image with this system, however, there is another system, microfluidic chambers, that may give you higher quality images.

Good Luck,

Maria

8.1

Posted by: AnonymousMarch 19, 2009, 11:56 AM

Dear Philippa, I have seen the video and read your comment only yesterday, but maybe this can help: at www.mattek.com, you can order 35mm or 50mm dishes with a partially glass bottom (coverslip that you can even take out afterwards and that is either uncoated or coated with collagen or poly-D-lysine). We have been using them on a regular basis for live cell imaging and they're very useful. From my side, I was wondering if you made any progress with scratching glass dishes, because I would like to do this for my particular experiment. Best regards, Katrien

8.2

Posted by: Katrien J.August 28, 2009, 5:01 AM

Hi Katrien,

Thank you for your suggestions. In answer to your question unfortunately I wasn't able to scratch the glass coverslips. Unfortunately I think it would be a case of having to get them engraved independently. I've been using the polystyrene cell culture dishes since and although maybe the optics aren't as good as they could I'm actually still able to obtain very good images after immunostaining with the inverted microscope. So if this is similar to what you plan to do it might be worth a try. Best of luck.

Philippa

8.2.1

Posted by: PhilippaSeptember 2, 2009, 3:29 AM

Hi Philippa, thanks for your answer. Would it be possible to let me know what magnification objective you use and what kind of structures you are looking at, just to have an idea if this might work for me as well?
Thank you and best regards, katrien

8.2.1.1

Posted by: Katrien J.September 2, 2009, 3:38 AM

Of course: I work with a 20x magnification and take a series of consecutive images of the neurites that have extended into the side compartments in order to reconstruct the whole image and assess neurite outgrowth before and after a treatment. I would prefer to use 10x magnification but we don't have the lens for it. Hope that helps. Philippa

8.2.1.1.1

Posted by: PhilippaSeptember 2, 2009, 12:59 PM

Any suggestions would be really appreciated.

Best regards, Philippa

Posted by: Philippa M.March 19, 2009, 8:12 AM

Any suggestions would be really appreciated.

Best regards, Philippa

Posted by: Philippa M.March 19, 2009, 2:04 PM

Hi,

Could you please elaborate a bit more on the DRG dissection and how you get to 100,000 cells please

Thanks,

Gustavo Ayala

R. Clarence and Irene H Fullbright Chair in Pathology

Professor

Baylor College of Medcine

Posted by: G. AyalaMarch 24, 2009, 4:58 PM

hi,

thank you for the presentation,

your novel model for preparation of drg neurons seem to be very efficient.

can you please elaborate how did you get the neurons and how old was the rat fetus?

thanks ahead,

Amit Moran,

moranamit@gmail.com

Posted by: moran amitMarch 30, 2009, 11:58 PM

It is very interesting. I am wondering regarding the use of NGF. You have used Recombinant human beta NGF. Is there any special reason you used human NGF? or is it ok that we can use rat NGF?

Posted by: AnandJune 23, 2009, 3:16 PM

Hi Anand-

We prefer to use the recombinant human NGF but it is certainly okay to use rat NGF.

Good Luck,
Maria

13.1

Posted by: AnonymousJune 30, 2009, 12:51 PM

Thanks for your presentation. Have you ever tried cultivating hippocampal neurons? If it 's, what did you coated with your dish, collagen or poly-l-lysine?

Regards

Mei

Posted by: MeiAugust 18, 2009, 4:35 PM

Hi Mei-

Sorry, we have never cultivated hippocampal neurons using this system. There is a paper, Ivins et al., 1998, that uses a modified compartmented culture system that you may find helpful.

Good Luck,
Maria

14.1

Posted by: AnonymousAugust 21, 2009, 1:39 PM

Dear colleges, thank you very much for nice performance and demonstration of this method!
I have a question. How do you think could I use this compartmented culture to investigated axonal degeneration. If I will add a substance under the investigation to axonal part of chamber, how could I be sure that this compound does not penetrate to cell body part?
Thank you very much for answer beforehand and good luck in your future experiment!!!

Posted by: Liudmila E.September 28, 2009, 9:17 AM

Hi Lula-
If set up properly (no leakage) these chambers are fluidically isolated.

Good Luck,
Maria

15.1

Posted by: AnonymousJanuary 13, 2010, 2:54 PM

Ok, how it could be isolated?: side parts and middle parts fluidically, if even neurites can pass through this barrier. That means that compounds (chemical substance) can do it also. am I wrong?

15.1.1

Posted by: AnonymousJanuary 14, 2010, 4:49 AM

Hi Maria,
I will like to confirm the concentration of collagen coating that you use have on the protocol (0,71mg/mL diluted in 0,001N HCl). In our lab we use collagen coating for cultures and the concentration is very less.
Regards
AleMor�n

Posted by: AleMoranSeptember 28, 2009, 1:09 PM

Hi Alemor-
Yes, that is the correct concentration of collagen.
Good Luck,
Maria

16.1

Posted by: AnonymousJanuary 13, 2010, 2:39 PM

Thanks Maria,
I would like to ask you a new cuestion. Did you cut de needle to avoid de sharp point?
Regards

16.1.1

Posted by: aleMarch 30, 2010, 11:29 AM

Hi-
The adapter that we use has a blunt end. It's a 23 gauge luer stub adapter (Fisher cat #427565).
Good Luck,
Maria

16.1.1.1

Posted by: AnonymousMarch 30, 2010, 2:55 PM

Hi, It is quite interesting. I need some help in isolating DRG. I tried. But not able to identify them. Can you pls help me.
Thanks in advance.

Posted by: anandOctober 26, 2009, 3:44 PM

Hi Anand-
The DRGs are located along each side of the spinal cord. In the E15 rats, the DRGs are clearly visible as ganglia along the spinal cord. At older ages the DRGs are encased within the vertebrae.
Good Luck,
Maria

17.1

Posted by: AnonymousJanuary 13, 2010, 2:42 PM

Hi,
Great presentation!
I was wondering, what antibodies did you use for the IHC images?

Thanks,
Amy

Posted by: AnonymousDecember 1, 2009, 8:43 PM

Hi,

many thanks for posting this really helpful video. When you apply the grease on the divider do you work under a hood? If not, how do you maintain sterility? I have tried working outside a hood and I have had problem of contamination (all solutions, and tools have been either filtered or autoclaved)

Kind regards,

Ale

Posted by: AlessandroJanuary 13, 2010, 11:31 AM

Hi Ale-
It would be best if you set up the cultures in a dissecting hood if you are having problems with contamination. We use UV to sterilize the area but the hood that we work under has no air flow and is therefore not a completely sterile environment.

Good Luck,
Maria

19.1

Posted by: AnonymousJanuary 13, 2010, 2:51 PM

Hi
Beautiful work! And I have been tried to constructed the compartmented cultures for many times but failed .I don't know why. I wonder how do you get to 100,000 cells .Could you please elaborate a bit more on the DRG dissection .In my compartmented cultures ,there are only a few axons across the barrier and get to the distal compartment.I have tried it for many times but the results are the same.I guss if the cell differentiation is not good enough or something wrong with the applying grease.I have no idea .So i think if you can give me your email and I have many problems to ask you.My email is jjdongch@gmail .com. I am looking forward to your reply .Thank you!

Posted by: jingjing d.May 17, 2012, 7:39 AM

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Date Published	10/17/2008
JoVE Section	General
JoVE Issue	20
Comments	18 Comments
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DOI	doi:10.3791/951

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隔间文化背根神经节神经元的制备及维修

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Cite this Article: 隔间文化背根神经节神经元的制备及维修

Abstract: 隔间文化背根神经节神经元的制备及维修

Protocol: 隔间文化背根神经节神经元的制备及维修

Discussion: 隔间文化背根神经节神经元的制备及维修

Disclosures: 隔间文化背根神经节神经元的制备及维修

Acknowledgements: 隔间文化背根神经节神经元的制备及维修

Materials: 隔间文化背根神经节神经元的制备及维修

References: 隔间文化背根神经节神经元的制备及维修

Ask the Author: 隔间文化背根神经节神经元的制备及维修

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