Compartmented 문화의 도설 루트 신경의 뉴런의 준비 및 유지 관리

Published 10/17/2008
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Summary

여기서 우리는 지느러미 루트 신경의 culturing 감각 뉴런에 compartmented 회의소 준​​비 및 유지 관리의 기법을 설명합니다.

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

뉴런은 멀리 대상 - 파생 성장 요소의 연결을 생존과 기능에 필요한 대상 조직을 신경을 분포시키다하는 세포 본체에서 제거됩니다 axonal 프로세스를 확장합니다. Neurotrophins은 특히 감각 뉴런을 innervating의 생존과 분화를 유지하기 위해 필요하지만, 이러한 목표 - 파생 neurotrophins은 innervating 뉴런의 세포 기관에 의사 소통 방법의 문제는 30 년 넘게 적극적인 연구 영역을했습니다. neurotrophin 신호가 세포 신체에 도달하는 방법 중 가장 일반적으로 인정 모델은 신호가 축삭을 따라 retrogradely이 신호를 운반 endosomes 것을 제안합니다. 역행 수송을 연구하기 위해, 문화 시스템은 원래 세포 시신은 axons으로부터 격리하는 로버트 Campenot에 의해 고안되었다. culturing 감각 뉴런의 recapitulates 대상 - 파생 neurotrophins의 생체내 다음 릴리스에서 발생하는 신경 세포 단말기의 선택적 자극에 대해 이러한 compartmented 회의소 준​​비의 기술. 장거리 미세 소관에 의존 역행 수송을 필요로 역행 신호 이벤트가 neurodegenerative 장애의 치료에 중요한 의미를했습니다.

Protocol

시약의 준비

  1. 콜라겐 코팅 : 콜라겐 코트 P35 조직 문화 접시 37 오븐에 장소 ° 디바이더를 기름 치는 것 이전 2 일간 C. 콜라겐의 최종 농도는 0.71 MG / 0.001 N HCL에 희석 ML에서해야합니다. 그런 다음, 접시 당 혼합물의 1 ML를 추가합니다.
  2. 그리스 로더 : 그리스 로더를 작성하기 위해, 60mL 주사기가 처음 코닝 진공 기름으로 가득해야합니다. 45 분 동안 압력솥 후, 그리스 로더를 작성 호일에 포장하고 주사기를 사용합니다.
  3. 테플론 디바이더 : 디바이더는 각 실험 후 다시 사용할 수 있지만 먼저 제대로 청소해야합니다. 접시에서 분배기를 제거 2 일 황산에 남아있는 기름과 장소를 모두 닦아. 산성에서 제거한 후, 물 3 배, 종기와 린스 20 분, 건조 20 분 유리 p100 페트리 접시와 압력솥의 자리를 수 있습니다.
  4. N2 - 메틸 셀룰로오스 : 메틸 셀룰로오스의 1.5g을 올리고 돛을 500mL 병에 넣습니다. 저어 표시줄을 추가하고 건조에 20 분 동안 그것을 압력솥 (이 시점에서 모든 작업을 살균해야합니다.) 다음 혈청 무료 미디어 (N 2) 500 MLS를 추가하고, 그것이 해소 때까지 차가운 방에 저어. -20 ° C. 50 ML conicals 및 동결로 나누어지는 -20 ° C.에서 일하는 주식, 1mL 튜브에 50 ML conicals 중 하나 나누어지는 및 동결에 대한
  5. DRG 미디어 : DMEM, 5 % 히트 inactivated 말 혈청,, 1 %의 페니실린 스트렙토 마이신.
  6. 100ng/mL DRGN 미디어 : 모두 신경 성장 인자 (NGF)와 두뇌 파생 neurotrophic 요인 (BDNF)의 주식 농도는 1mg/mL입니다. DRG 미디어에 neurotrophins 1:10,000 각 희석. 문화는 NGF 혼자 재배 수 있으며, 이것이 문화에서 살아남기 뉴런의 보완을 바꿀지도 모르겠어.

    참고 : 필요한 경우, (사이 토신 arabinoside) AraC의 농도가 1uM 및 0.3uM의 최종 농도에 사용됩니다. 이것은 Schwann 세포와 다른 glia의 성장을 억제합니다.

  7. 10ng/mL DRGN 미디어 : DRG 미디어 100ng/mL DRGN 미디어 (1시 10분)을 희석.

    그림 1


    설정에 필요한 그림 1. 도구

(해부하기 전에 1-2일이 과정을 시작합니다) compartmented 회의소 설정

  1. 밖으로 운동과 함께 콜라겐 코팅 P35 접시의 중앙에 흠집을 만듭니다.
  2. 처음의 중간에 N2 - 메틸 셀룰로오스의 플레이스 30ul. 분할기가 기름칠 때까지 따로 요리를 설정합니다.
  3. 그리스 로더에 23 게이지 luer 스텁 어댑터를 연결합니다. 그립 테플론 90 ° 각도 hemostats 한 쌍의로 구분선하고 현미경 아래에 직면하고있는 분할기와 함께 그것이 평평하다. 그리스는 그리스의 연속 라인 (그림 참조)가 너무 때마다 어댑터는 어댑터가 이전 단계에서 그리스에 삽입하는 새로운 출발점에 배치되었는지 확인하고있는 분배기를 추적. 그리스가 전체 구분선에 적용되면 N2 - 메틸 셀룰로오스는 중간 구획 이상하므로, 거꾸로 준비 P35 요리 중 하나를 설정하고 그것을 놓으십시오. 핀셋 한 쌍의와 접시의 바닥에 아래 버튼을 누르십시오. 네 모퉁이 ( "X"로 다이어그램에 표시된 왼쪽 상단, 오른쪽 아래, 오른쪽, 왼쪽 아래)에있는 구분선의 안쪽에 언론에 있는지 확인하십시오.

    그림 2

    그림 2 : 구분선을 기름 치는 것 단계

    참고 : 그것을 그리스의 음식과 완벽한 도장을 만드는 있도록 단단히 정도로 언론에 중요하지만, 너무 많은 압력이 추가 경우, axons는 측면 구획에 교차하지 않습니다.

    , hemostats를 들고 그것을 뒤집 및 구분선을 unclamp. 마지막으로, 단단히 중간 구역의 하단에 초점 현미경 첨부 분할기와 함께 요리를 놓으십시오. 그리스 로더로 세포가 중간 구획에 배치되면 그들이 누설 수 있도록 작은 장벽 (0.25 cm)를 확인하십시오.
  4. 여러 문화, 측면 구획의 각 장소 DRG 미디어와 전지가 유지됩니다되는 배양기에서 위치를 설정하는 데 후. 문화가 몇 시간 동안 앉아 후 누출을 확인할 수 있습니다. 미디어가 중간 구획으로 유출있다면, 다음 문화 사용할 수 없습니다.

    참고 : 여러가 새는되므로 먼저 기술을 배우고, 그것이, 실험에 필요한 이상의 문화를 설정하는 것이 중요합니다.

    그림 3

    그림 3 : "좋은 VS 새서 '문화

compartmented 문화의 유지 DRG 뉴런

  1. 1 일 : 100ng/mL DRGN 미디어 + AraC과 측면 구획에서 미디어를 DRG 교체합니다. 절개를 수행하고 중심 구획 (100000 셀)에 세포를 추가합니다.
  2. 일 2 : 미디어 센터 구획과 그리스의 장벽과 교류를 통해 흐르는 유체 때까지 테플론 분할기의 외부 10ng/mL 미디어 + AraC를 추가합니다.
  3. 3 일 : 10ng/mL DRGN는 AraC를 생략로 AraC와 서라운드를 생략 100ng/mL DRGN하는 측면 구획에서 미디어를 교체하십시오.
  4. 일 6 : 1ng/mL + AraC 및 DRG 미디어 + AraC으로 둘러싸고있는 측면 구획에서 미디어를 교체하십시오.
  5. 일 9 : 실험에 사용.


    그림 4

    그림 4 : 세포 기관과 말초 axons의 IHC 이미지



    참고 : 미디어를 변경하면, 그것은 각각의 측면 구역의 상단에서 액체를 대기음하는 것이 중요합니다. 또한, 오직 서라운드에서 중간 구획 자체에서 미디어를 변경할 수없고, 그것이 중심에 그리스의 장벽을 통해 흘러가게 마십시오.

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Discussion

이 비디오에서는, 우리는 culturing의 DRG의 뉴런에 사용하기 위해 compartmented 회의소를 준비하고 유지하는 방법을 증명하고있다. 제대로 완료,이 시스템은 neurotrophins 오래 axons에 걸쳐 신호를하는 메커니즘을 연구하기 위해 축삭에서 세포 기관의 분리 수 있습니다. 구획 사이에 유체 절연이 있기 때문에, 그것은 다른 구획에 영향을받지 않고 한 구역의 선택적 자극이나 치료 수 있습니다. Compartmented 챔버 문화는 우수 자궁 경부 신경, 망막 신경절의 뉴런과 뉴런에서 대뇌 피질의 교감 신경 등 다른 세포 유형을 지원할 수 있습니다. neurotrophin 신호 전달의 공간적 이해 neurodegenerative 장애의 치료에 새로운 통찰력을 제공할 수 있습니다. 알츠하이머 병, 헌팅턴의 질환과 운동 신경 질환을 포함한 몇몇 neurodegenerative 장애는, axonal 교통의 결함과 관련 있습니다. 최근 연구 microfluidic 챔버 대신 이러한 compartmented 회의소를 사용했습니다. 4,5 microfluidic 챔버 스는 이미지 분석을위한 몇 가지 장점이 있습니다.

이전 연구는 축삭과 세포 바디 구획 1,3,6 사이의 확산을 방지하기 위해 이들 문화의 능력을 테스트했습니다. 이것은 쉽게 하나의 구획 이러한 trypan 파란색으로 염색의 낮은 농도를 추가하고, 염료의 확산 찾는하여 테스트할 수 있습니다. 24 시간 이내에 볼 수 거의 또는 전혀 보급이 있어야합니다.

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Acknowledgements

우리는 도움이 토론 Katharina Cosker과 스테파니 코세인 감사하고 싶습니다.

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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