コンパートメント文化の後根神経節ニューロンの準備とメンテナンス

Published 10/17/2008
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Summary

ここでは、後根神経節の培養感覚ニューロンについてはコンパートメント室を準備し、維持するテクニックについて説明します。

Cite this Article

Copy Citation

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).

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Abstract

ニューロンは、遠くのターゲット由来の成長因子がニューロンの生存と機能に必要な標的組織を、支配するために細胞体から削除されている軸索のプロセスを拡張します。ニューロトロフィンは、特に感覚神経の神経支配の生存と分化を維持することが義務付けられていますが、これらの標的由来神経栄養因子は神経支配ニューロンの細胞体への通信方法の質問は、30年以上にわたり活発な研究分野となっている。ニューロトロフィンのシグナルが細胞体に到達する方法の最も一般的に受け入れられているモデルは、シグナルが軸索に沿って逆行性にこの信号を運ぶエンドソームことを提案している。逆行性輸送を研究するために、培養系は、もともと細胞体がそれらの軸索から隔離されているロバートCampenot、によって考案されました。培養感覚ニューロンの反復するターゲット由来神経栄養因子のin vivoで、次のリリースで発生するニューロンの端末の選択刺激のためにこれらのコンパートメントチャンバーの準備のテクニック。長距離微小管依存性の逆行輸送を必要とする逆行性シグナル伝達事象は、神経変性疾患の治療のために重要な意味を持っている。

Protocol

試薬の調製

  1. コラーゲンコーティング :オーブンでコラーゲンコートp35の組織培養プレートと場所37℃で分周器を給脂する前に2日間C。コラーゲンの最終濃度は0.001 N塩酸で希釈した0.71 mg / mlのになっているはず。その後、プレート当たり混合物を1 mlを加える。
  2. グリースローダー :グリースローダーを埋めるために、60mlの注射器が最初にコーニングの真空グリースを封入する必要があります。グリースローダーを埋めるために注射器を使用して、45分間オートクレーブしてホイルに包んでと。
  3. テフロン仕切り :分周器をそれぞれの実験後に再使用することができますが、最初の適切に洗浄する必要があります。プレートから分周器を取り外す、2日間硫酸の残りのグリースと場所のすべてをふき取ってください。酸から削除した後、水3X、20分間沸騰とリンス、、20分間ガラスP100ペトリ皿とオートクレーブ内の場所を乾燥することができます。
  4. N2 -メチルセルロース :メチルセルロースの1.5グラムを秤量し、500ミリリットル瓶に入れます。攪拌棒を追加し、乾燥した上で20分間オ​​ートクレーブ(この時点から、すべての作業は無菌でなければならない)。次に、無血清培地(N 2)、500 MLSを追加し、それが溶けるまで寒い部屋でかき混ぜる。 -20℃で50mLのconicalsと凍結に分注し-20℃で作業用ストック、1mLのチューブに50 mLのconicalsの一分注して凍結するための
  5. DRGメディア :DMEM、5%熱不活化ウマ血清、及び1%ペニシリンストレプトマイシン。
  6. 100ng/mlのDRGNメディア:両方の神経成長因子(NGF)及び脳由来神経栄養因子(BDNF)の株式の濃度は1mg/mLです。 DRGのメディアへのニューロトロフィン1:10,000のそれぞれを希釈する。培養物はNGF単独で成長させることができる、これは文化の中で生き残るニューロンの補数を変更します。

    :必要なときは、(シトシンアラビノシド)AraCの濃度が1uMと0.3uMの最終濃度で使用されている。これは、シュワン細胞や他のグリア細胞の成長を抑制します。

  7. 10ng/mL DRGNメディア :DRGのメディアで100ng/mlのDRGNメディア(1:10)希釈。

    図1


    セットアップに必要な図1。ツール

コンパートメントチャンバーのセットアップ(解剖前に1〜2日、このプロセスを開始する)

  1. 外側への動きとコラーゲンコーティングしたp35の皿の中央にスクラッチを行う。
  2. スクラッチの真ん中でN2 -メチルセルロースの30ul場所。分周器が油を塗ったされるまで、脇に料理を設定します。
  3. グリースのローダに23ゲージルアースタブアダプタを取り付けます。グリップテフロンの90 °の角度止血のペアで分周器とは、顕微鏡下で上向きに分周器とそれを寝かせて。グリースは、それぞれの時間は、アダプタが新しい出発点に置かれているグリースの連続的なラインが存在するようにアダプタが前のステップからグリースに挿入されていることを確認すると分周器をトレース(図を参照)。グリスが全体の分周器に適用されるとN2 -メチルセルロースは、中央の区画を超えているので、上下逆さまに準備したp35の料理のいずれかをオンにして置きます。ピンセットで皿の底を下に押してください。四隅("X"によって図に示されている左上、右下、右上、左下、)で分周器の内側に押してください。

    図2

    図2:分周器を給脂する手順

    :これは、グリースが料理と完全なシールを作るが、過剰な圧力が付加されている場合、軸索は側の区画にまたがることがないようにしっかりと十分に押すことが重要です。

    、止血を拾うそれを裏返して分周器をアンクランプ。最後に、しっかりと中間のコンパートメントの底部に焦点を当てて顕微鏡下に取り付けられた分圧器を使って皿を置く。グリースのローダーで、細胞が中央コンパートメント内に配置されると、彼らが漏れないことができるように小さな障壁(0.25 cm)を行う。
  4. 複数の文化、サイド区画のそれぞれの場所のDRGのメディアと細胞が維持されるのインキュベーター内で場所を設定した後。文化は数時間放置し、漏れをチェックすることができます。メディアが真ん中のコンパートメントに流出している場合には、文化は使用不能です。

    :いくつかが漏れになるので最初にこのテクニックを学ぶとき、それは、実験に必要な数よりも多くの文化を設定することが重要です。

    図3

    図3:"グッドVS漏れ"文化

コンパートメントの文化の維持DRGニューロン

  1. 1日目 :100ng/mlのDRGNメディア+ AraCのある側のコンパートメントにDRGメディアを交換してください。解剖を行い、中央の区画(100,000セル)にセルを追加。
  2. 2日目 :メディアセンターのコンパートメントを持つグリースの障壁と交換流体を介して流れるまで、テフロンの分周器の外側に10ng/mLメディア+ AraCのを追加。
  3. 3日目 :10ng/mL DRGNはAraCのを省略してAraCのとサラウンドを省略100ng/mlのDRGNする側のコンパートメントでメディアを交換してください。
  4. 6日目 :1ng/mL + AraCのとDRGメディア+ AraCのあるサラウンドにサイドコンパートメント内のメディアを交換してください。
  5. 9日目 :実験のために使用して下さい。


    図4

    図4:細胞体と軸索遠位のIHC画像



    注:メディアを変更する場合、それは各サイド区画の上部から液体を吸引することが重要です。また、唯一のサラウンドから、中間のコンパートメント自体からメディアを変更していない、そして中央にグリスの障壁を越えて、流れてみましょうね。

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Discussion

このビデオでは、我々は培養DRGニューロンにおける使用のためにコンパートメント室を準備し、維持する方法を示している。正しく行えば、このシステムは、ニューロトロフィンは、長い軸索を介して通知されるメカニズムを研究するために、軸索から細胞体を分離することができます。コンパートメントの間に流体分離があるので、それは他のコンパートメントが影響を受けることなく、一区画の選択的な刺激や治療が可能になります。コンパートメントチャンバー培養は上頚神経節から交感神経ニューロン、網膜神経節ニューロン、および皮質ニューロンを含む他の細胞型をサポートできます。ニューロトロフィンのシグナル伝達の空間的な理解は、神経変性疾患の治療に新たな洞察を提供することがあります。アルツハイマー病、ハンチントン病や運動ニューロン病を含むいくつかの神経変性疾患は、軸索輸送の欠陥に関連付けられています。最近の研究では、マイクロ流体チャンバーの代わりにこれらのコンパートメントチャンバーを使用している。 4,5マイクロ流体チャンバーは、画像解析のためのいくつかの利点があります。

先行研究では、軸索と細胞体のコンパートメント1,3,6の間の拡散を防ぐために、これらの培養物の能力をテストしている。これは簡単にだけ一区画にトリパンブルーなどの色素の低濃度を加えることによってテストされ、染料の拡散を探すことができます。 24時間以内可視ほとんど、またはまったく拡散があるはずです。

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Acknowledgements

我々は、有用な議論をカタリーナCoskerとステファニーCourchesneに感謝します。

Materials

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

DOWNLOAD MATERIALS LIST

References

  1. Campenot, R. B. Independent Control of the Local Environment of Somas and Neurites. Methods in Enzymology. 58, 302-307 (1979).
  2. Watson, F. L., et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience. 4, 981-988 (2001).
  3. Heerssen, H. M., et al. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nature Neuroscience. 7, 596-603 (2004).
  4. Taylor, A. M., et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods. 2, 599-605 (2006).
  5. Park, J. W., et al. Microfluidic culture platform for neuroscience research. Nat Protoc. 4, 2128-2136 (2006).
  6. Ure, D. R., et al. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neuroscience. 4, 1282-1290 (1997).

Comments

49 Comments

  1. Hi 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, dŒs it provide a space through which the axons can pass, do the cell bodies settle on the collagen surface. Regards Paul

    Reply
    Posted by: Anonymous
    November 19, 2008 - 7:27 AM
  2. 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

    Reply
    Posted by: Anonymous
    December 15, 2008 - 3:06 PM
  3. Hi, your method looks simple and powerfull. It's possible to make an immuno staining without to remove the teflon divider? DŒs the divider cause troubles during immunofluorescence staining for example? Regards Giuseppe 

    Reply
    Posted by: Anonymous
    December 13, 2008 - 7:35 AM
  4. 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

    Reply
    Posted by: Anonymous
    December 15, 2008 - 3:52 PM
  5. 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.

    Reply
    Posted by: Anonymous
    October 22, 2010 - 7:01 PM
  6. Hi Supraja-
    Yes, only remove the divider at the end of the immunostaining. Everything else is done exactly the same.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    October 25, 2010 - 3:42 PM
  7. Hi, beautiful work, congratulations! Have you tried to cultivate adult DRG also? Best regards and good luck! Otilia

    Reply
    Posted by: Anonymous
    February 5, 2009 - 6:34 AM
  8. Hi Otilia- Thank you for your comment.  We have not tried cultivating adult DRGs in this system.   Good Luck, Maria

    Reply
    Posted by: Anonymous
    March 19, 2009 - 12:03 PM
  9. 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

    Reply
    Posted by: Anonymous
    September 2, 2009 - 1:02 PM
  10. Thanks a lot, Philippa!

    Reply
    Posted by: Anonymous
    January 18, 2010 - 4:08 PM
  11. 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

    Reply
    Posted by: Anonymous
    February 11, 2009 - 3:39 PM
  12. 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 Yi

    Reply
    Posted by: Anonymous
    March 5, 2009 - 3:52 PM
  13. Hi 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 dŒsn'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

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 7:25 AM
  14. 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  

    Reply
    Posted by: Anonymous
    March 19, 2009 - 11:56 AM
  15. 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

    Reply
    Posted by: Katrien J.
    August 28, 2009 - 5:01 AM
  16. 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

    Reply
    Posted by: Anonymous
    September 2, 2009 - 3:29 AM
  17. 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

    Reply
    Posted by: Katrien J.
    September 2, 2009 - 3:38 AM
  18. 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

    Reply
    Posted by: Anonymous
    September 2, 2009 - 12:59 PM
  19. Hi 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 dŒsn'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

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 8:12 AM
  20. Hi 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 dŒsn'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

    Reply
    Posted by: Philippa M.
    March 19, 2009 - 2:04 PM
  21. 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  

    Reply
    Posted by: Anonymous
    March 24, 2009 - 4:58 PM
  22. 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    

    Reply
    Posted by: Anonymous
    March 30, 2009 - 11:58 PM
  23. 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?

    Reply
    Posted by: Anonymous
    June 23, 2009 - 3:16 PM
  24. Hi Anand-

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

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    June 30, 2009 - 12:51 PM
  25. 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

    Reply
    Posted by: Anonymous
    August 18, 2009 - 4:35 PM
  26. 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

    Reply
    Posted by: Anonymous
    August 21, 2009 - 1:39 PM
  27. 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 dŒs not penetrate to cell body part?
    Thank you very much for answer beforehand and good luck in your future experiment!!!

    Reply
    Posted by: Liudmila E.
    September 28, 2009 - 9:17 AM
  28. Hi Lula-
    If set up properly (no leakage) these chambers are fluidically isolated.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:54 PM
  29. 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?

    Reply
    Posted by: Anonymous
    January 14, 2010 - 4:49 AM
  30. 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
    AleMorán

    Reply
    Posted by: Anonymous
    September 28, 2009 - 1:09 PM
  31. Hi Alemor-
    Yes, that is the correct concentration of collagen.
    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:39 PM
  32. Thanks Maria,
    I would like to ask you a new cuestion. Did you cut de needle to avoid de sharp point?
    Regards

    Reply
    Posted by: Anonymous
    March 30, 2010 - 11:29 AM
  33. Hi-
    The adapter that we use has a blunt end. It's a ²3 gauge luer stub adapter (Fisher cat #4²7565).
    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    March 30, 2010 - 2:55 PM
  34. 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.

    Reply
    Posted by: Anonymous
    October 26, 2009 - 3:44 PM
  35. 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

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:42 PM
  36. Hi,
    Great presentation!
    I was wondering, what antibodies did you use for the IHC images?

    Thanks,
    Amy

    Reply
    Posted by: Amy M.
    December 1, 2009 - 8:43 PM
  37. 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

    Reply
    Posted by: Anonymous
    January 13, 2010 - 11:31 AM
  38. 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

    Reply
    Posted by: Anonymous
    January 13, 2010 - 2:51 PM
  39. 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!

    Reply
    Posted by: jingjing d.
    May 17, 2012 - 7:39 AM
  40. Hi!, excellent video!. We are reproducing this system, but now, we have problem with collagen coating. It does not gelled 3 or 5 days after!, in fact, it does not change. We´re using the same collagen (BD Bioscience ,354249) and your final concentration (0.71mg/ml diluted in 0.001N HCl). What recommendations do you have?.

    Kind Regards!

    Reply
    Posted by: DIANA M.
    January 9, 2014 - 1:00 PM
  41. Hi Diana-
    The plates need to be put in a dry oven at 37 degrees for 3 days. Are you doing that? Also, the collagen doesn't gel, it dries completely. Good luck and feel free to email me with any additional questions. maria_pazyra@dfci.harvard.edu

    Reply
    Posted by: Anonymous
    January 9, 2014 - 1:29 PM
  42. Great article. What are the product numbers and companies for methylcellulose and N2 serum free media?

    Reply
    Posted by: Eric W.
    June 17, 2014 - 3:04 PM
  43. Hi Eric- The methylcellulose we currently use is from Xenex (catalog # E4M) and the serum free media is just plain DMEM. Good Luck, Maria

    Reply
    Posted by: Anonymous
    June 17, 2014 - 3:21 PM
  44. Hi, Maria
    Your article and video in jove helped our research a lot, excellent!
    I have one question. Are you still using N2-MC? Please let me know, if there is better one that can prevents grease from closing grooves.
    Thank you
    S
    Thank you

    Reply
    Posted by: Shingo K.
    October 13, 2015 - 11:31 AM
  45. Hi Shingo-
    I'm not sure I understand your question. Are your axons not growing through the grooves? The N2 is used to help the axons slide under the grease and we still use the Xenex brand. If your axons are not growing through it is probably more about how much pressure you are applying when you press the dish to the teflon divider.

    Good Luck,
    Maria

    Reply
    Posted by: Anonymous
    October 13, 2015 - 11:54 AM
  46. Hi Maria-
    Thanks for your quick reply.
    Axons do grow well into the next chamber with N2-MC, but not through all the grooves I made (~50%). I am wondering if there is other potential substance (i.e., one with higher viscosity) that I can try...
    I am expecting more... or do you think ~50% is OK ?
    shingo

    Reply
    Posted by: Shingo K.
    October 13, 2015 - 1:53 PM
  47. Shingo-
    My suggestion would be to add more N2 to cover more of the grooves. I don't know of any other substance that would work. 50% is actually pretty good.

    Best,
    Maria

    Reply
    Posted by: Anonymous
    October 13, 2015 - 2:09 PM
  48. Hi Maria,

    Thank you for publishing this protocol. I have been trying to grow cortical neurons in a custom made Teflon divider (1.5mm width of central compartment and 0.8mm height of divider), without much success. The cell bodies leak in the side compartments.
    I have coated the dish with poly-d-lysine instead of collagen do you think this can affect the chamber set up? Have you ever tried to culture cortical neurons in this chamber or know someone who has? I'm thinking that perhaps the cell body of cortical neurons is smaller than DRGs and this could cause leakage, what do you think?

    Regarding the methylcellulose step. I diluted it in Neurobasal (already supplemented with Pen Strep and Glutamax). I've added it on the middle of the scratched region and then applied the divider with grease in the same way as you explain in the video.I removed it before adding the cells but I did not let it dry. Are you supposed to leave the drop medium with methylcellulose when cells are added? How long do the cells need before they attach to the dish completely?

    Any advice on how to improve my method would be highly appreciated.

    Thanks a lot in advance,

    Anna

    Reply
    Posted by: University of Aberdeen .
    April 5, 2016 - 2:05 PM
  49. Hi Anna-
    We have never cultured cortical neurons in this system before but I do think that the collagen or matrigel (that's what we use now) is essential. I don't think the size of the cells is a factor. It's probably more about the grease. And mastering that only comes with lots and lots of practice. Are you using these for biochemistry? If you are using them for staining or imaging you should look into microfluidic chambers. We use those in the lab too. We leave the methylcellulose in the center compartment so no need to remove it before plating the cells....and the cells should attach within a couple of hours. Good luck and please feel free to email me with any further questions. maria_pazyra@dfci.harvard.edu

    Best,
    Maria

    Reply
    Posted by: Anonymous
    April 5, 2016 - 3:34 PM

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